Comparative study of the efficacy of potassium magnesium L-, D- and DL-aspartate stereoisomers in overcoming digoxin- and furos

ARTICLE

Auteur(s) :, Igor N Iezhitsa^1,*, Alexander A Spasov¹, Natalia V Zhuravleva¹, Maxim K Sinolitskii², Sergey P Voronin²

¹Volgograd State Medical University, Research Institute of Pharmacology, 1 Pavshikh Bortsov Sq., Volgograd, 400131, Russia
²“BioAmid”, 27 Mezhdunarodnaya St., Saratov, 410033, Russia

Introduction

Mg deficiency may arise together with, and contribute to, the persistence of K deficiency and cases of Mg deficiency accompanying Mg-dependent or -independent K deficiency are not uncommon among the general population. Both may develop as a result of insufficient dietary intake, abnormal gastrointestinal loss or abnormal renal loss. K and Mg deficiencies are found in patients with chronic alcoholism [3-7], in the elderly [8], during therapy with some kind of drugs (such as amphotericin B [9], gentamicin [10, 11], cisplatin [12, 13], cardiac glycosides (digoxin) [14-19], distal (thiazides) and loop (furosemide) diuretics [20-24]), severe diarrhea [25-28], malnutrition [28], cardiac diseases [29-32], diabetes mellitus [33, 34], genetic forms of renal potassium and magnesium wasting (Gitelman’s and Bartter’s syndromes) [35, 36], etc. Of special interest is the Mg and K status in cardiac diseases [29-32]. Hypomagnesemia and hypopotassemia complicate a variety of cardiovascular diseases, including congestive chronic heart failure, myocardial ischemia, acute myocardial infarction with sudden death, and recurrent and therapy-unresponsive arrhythmias, etc [29-32]. K and Mg deficiencies are often coexistent and pathophysiologically related in patients with heart disease, and, in some cases, K deficiency cannot be treated without correction of concomitant Mg deficiency [29]. So K and Mg depletion are commonly concomitant, and simultaneous repletion of both ions would be both logical and effective in various diseased states. Various K,Mg salts allowing simultaneous elimination of the deficiency of Mg and K are described in the literature. They are K, Mg aspartate [37-42], K,Mg nicotinate [37], K,Mg citrate [43, 44], K,Mg glutamate [38], K,Mg nicotinylaspartate [45, 46] etc.

Among these salts K,Mg aspartate has been the most studied. For the first time, the original combination of K and Mg aspartates was proposed at the end of the 1950s by French scientist Henri Laborit and his scholars [47-52]. He showed that K,Mg aspartate could be used as adjuvant therapy in heart disease [47, 48]. Continued study through the early 1960s confirmed his research in clinical practice [53, 54]. In the 1960s, the German scientist Hans A Nieper put forward the disputable theory of “mineral transporters”, substances that he believed would increase the bioavailability of minerals in tissues and cells [45, 46, 55]. According to the Nieper’s conceptual model of “mineral transporters”, the aspartate-ion promotes the penetration of K and Mg through the cellular membranes and aspartate itself is metabolised [45, 46]. Unfortunately, while they sound plausible, there is essentially no published research that confirms or refutes them, and Nieper’s explanations are no longer accepted by the scientific community.

K,Mg aspartate is now the most prescribed among K,Mg salts [37-42]. It can be used as adjuvant therapy in ischaemic heart disease (in angina pectoris and conditions after myocardial infarction), prophylaxis and adjuvant therapy of cardiac arrhythmia (e.g. prevention of toxic symptoms during therapy with digoxin) [39-42]. K,Mg aspartate decreases risk of development of cardiac arrhythmia in ischaemic heart disease, improves tolerance of digoxin and replaces K and Mg loss during treatment with diuretic drugs. K,Mg aspartate can be used as an adjuvant therapy of electrolytic disturbances in alcoholism [56], because depletion of K and Mg appears to play a significant role in the ventricular tachyarrhythmias seen in alcoholic patients, as well as in patients treated with diuretics and/or patients with digitalis toxicity [32].

Widely used, K,Mg aspartate is synthesized from aspartic acid representing a racemic mix of L- and D-stereoisomers. It has been believed for a long time that amino acids are exclusively L-configuration in eukaryotes, especially in higher animals, and that the L-configuration of amino acid predominates in all known living systems [57]. D-amino acids are considered to be xenobiotic substances and not nutrients. Metabolism and utilisation of D- and L-aspartic acids differ. It is well known that L-aspartic acid is mainly absorbed by the small intestine using a specific amino acid transporter located at the brush border membrane [58]. L-Aspartic acid occupies an important position in the TCA cycle, urea cycle and nucleic acid formation pathway. D-Aspartic acid is as easily absorbed as L-amino acid at the small intestinal site using a sodium ion-dependent transport system on the brush border membrane of the intestinal epithelial cells [58]. D-Amino acids in the body are thought to be metabolized into α-oxoacids by D-amino acid oxidase or D-aspartic acid oxidase [59, 60]. Although most of the D-amino acids were oxidized by the liver, kidney and other tissues after intestinal absorption, it is possible for D-amino acids to pass through these enzymatic barriers to be distributed in various tissues throughout the mammalian body [58]. D-Aspartic acid has been found in various regions of the rat brain such as the cerebrum and diencephalon (pituitary gland and pineal gland), in rat peripheral organs such as the adrenal gland, testis and kidney, in human brain frontal cortex and cerebrospinal fluid [61-67]. The real relationship between D-amino acids and biological function or disease is not clear at this time [58].

Until recently, it was not clear whether D-aspartate was as safe as L-aspartate. A study by Schieber et al. [68] produced no evidence for subacute toxicity of orally fed D-aspartic acid (50 mg amino acid per kg of body weight for 28 days). Continuous feeding with D-aspartate did not increase renal excretion of this enantiomer, but in the serum, higher amounts (0.8-4.0 μmol/1) were determined in comparison to the control group (0.3-0.9 μmol/1) [68]. Unfortunately there are no comparative toxicity data of intravenously administered L-aspartate and D-aspartate. In addition, the above-mention studies were carried out with free amino acids but not their salts and consequently it is rather difficult to reach a conclusion concerning any toxicity and pharmacology of K,Mg D- and L-aspartates. On the other hand, differences in the metabolism and utilisation of D- and L-amino acids may have an effect on the pharmacological properties of K,Mg L- and D-aspartates, and what is more pharmacological doses of magnesium and potassium salts may induce toxicity, which differs according to the nature of the anions (e.g., it has been already shown for MgCl₂ and MgSO₄[69, 70]).

In our preliminary study [71] it was found that intravenously administered K,Mg L-aspartate exhibits greater activity compared to K,Mg D- and DL-aspartate on the strophanthin K, calcium chloride and aconitine-induced arrhythmia models. K,Mg L-aspartate resulted in a greater decrease in the incidence of arrhythmias, increased the time to onset of the first arrhythmia, decreased the percentage of rats that died and increased the duration of life after onset of the first arrhythmia in rats with arrhythmias induced by strophanthin K and calcium chloride as compared with K,Mg D-aspartate and K,Mg DL-aspartate [71]. K,Mg L-aspartate surpassed K,Mg D-aspartate and K,Mg DL-aspartate in parameters of acute toxicity (LD₅₀), effective dose (ED₅₀) and antiarrhythmic (therapeutic) ratio (LD₅₀/ED₅₀) in rats with aconitine-induced arrhythmia model [71] (table 1( Table 1 )).

So, the purpose of the present work was to study the effect of intravenously administered K,Mg L-aspartate in comparison with its D- and DL-stereoisomers on K and Mg restoration rate in plasma, erythrocytes and myocardium and to evaluate the urine excretion rate of amine nitrogen and Mg in digoxin and furosemide treated rats.
Table 1 Parameters of acute toxicity (LD₅₀), effective dose (ED₅₀) and antiarrhythmic ratio (LD₅₀/ED₅₀) of K,Mg L-, D- and DL-aspartate stereoisomers in rats with aconitine-induced arrhythmia model

Materials and methods

General

Digoxin- and furosemide-induced deficiencies of Mg and K

To induce Mg and K depletion, male rats, weighing 180-200 g, were given furosemide (1st series of experiments) and digoxin (2nd series of experiments) at doses of 30 mg/kg (i.p.) and 0.25 mg/kg (i.p.) daily for 14 days. After 14 days K,Mg L-, D- and DL-aspartates were administered with simultaneous furosemide and digoxin treating at a dose of 100 mg/kg (i.v.), that corresponds to 46.95 mg of Mg aspartate [i.e. Mg = 3.96 mg] and 53.05 mg of K aspartate [i.e. K = 12.12 mg] per kg bodyweight. Preliminary injections of K,Mg L-, D- and DL-aspartates solutions were dissolved in isotonic glucose in the ratio 1:2. Control animals were injected with isotonic glucose (control II). There was also a group of animals that received neither furosemide/digoxin, nor K,Mg aspartates or glucose – intact animals (control I).

In the present study, K,Mg L-, D- and DL-aspartates were administered intravenously. On the one hand, K,Mg aspartate injection is now widely used in treating and preventing cardiac disturbances caused by electrolytic disturbances, primarily a low K and Mg level (e.g. in the treatment with cardiac glycosides and diuretic drugs) [39-42]. On the other hand, the effect of intravenous K,Mg aspartates is more interesting than the effect of per oral K,Mg aspartates because orally administered aspartates can be metabolized by enterocytes or eliminated by first-pass hepatic effect. Expressed in other words, the level of free D-amino acids in mammals body can be regulated by D-amino acid oxidase and the greater part of D-amino acids absorbed cannot be further distributed to the body as a whole because they are trapped by the liver, like a barrier [58, 60]. If D-amino acids pass through the barrier (e.g., i.v. injected), they are transported throughout the whole body.

To study the evolution of Mg deficiency, erythrocyte and plasma Mg levels were measured before the beginning of the experiment, and after the first, second and third weeks of the experiment. To compare K and Mg deficiency correction rate in digoxin and furosemide treated rats after single intravenously administered K,Mg L-, D- and DL-aspartate stereoisomers, erythrocyte and plasma Mg contents were estimated after 3, 6 and 24 hours of K,Mg aspartate injection. Then after 24 hours, 5 rats from each group were killed by decapitation for myocardium Mg and K content and erythrocyte K levels determination. To compare the efficacy of three stereoisomers of K,Mg aspartate in overcoming furosemide/digoxin-induced hypokalemia and Mg loss in a one week course, 5 rats from each group were killed after 7 days for erythrocyte, plasma and myocardium Mg and K content determination. The experimental design for furosemide and digoxin experimental series was analogous (( figure 1 )).

To compare increasing urinary aspartate and Mg after single intravenously administered K,Mg L-, D- and DL- aspartate stereoisomers, the content of urine amine nitrogen and Mg after 4, 8, 12, 16, 20 and 24 hours was measured by colorimetric assay using the methods based on the staining reaction with ninhydrin [72] and thiazole yellow [73], respectively.

Drugs

Measurements

Isolation and preparation of plasma and erythrocytes [73]

Erythrocytes were washed three times by suspension in an iso-osmolar NaCl (0.9%) solution for Mg determination. The cells were washed by centrifugation at 1500 rpm for 7 min and aspiration of the supernatant. The erythrocyte suspension was lysed by the addition of demineralized water (0.5 mL erythrocyte suspension and 2.5 mL water). The lysate was prepared as for plasma.

Calibration curve. Calibration solution (1 mmol/L MgSO₄) from 0.2 till 1 mL were added in series of tubes, then demineralized water was added up to a volume 6 mL. One millilitre 2% hydroxylamine hydrochloride, 1 mL 0.075% thiazol yellow and 2 mL 1.5 N NaOH were then added. The colouring solution, in which 0.2 mL MgSO₄ was added, corresponded to the plasma Mg level of 0.4 mmol/L; solution containing 0.5 mL MgSO₄ corresponded to the level of 1 mmol/L, etc. [73].

Potassium and magnesium levels determination

Mg and K contents in heart and erythrocyte K levels were determined by flame atomic absorption spectroscopy (SF-115-M1 spectrometer, Russia) of samples previously ashed at 450 °C, until the weight was stable, in a furnace with increments of 1.5 °C/min from room temperature to 450 °C. Mineralization was continued at this temperature for 10-15 hours until ashes became a grey colour. Cooled to room temperature, ashed samples were then moistened with nitric acid (1:1) at 0.5 mL per sample, evaporated on a water bath and dried in the electric furnace with incrementing temperatures to 300 °C for 30 minutes. This operation was repeated 2-3 times. Mineralization was considered to be complete when ashes became a white colour. Ashed samples were then extracted with 2 mL solution of HCl (1:1), ashes were dissolved by heating on a water bath, evaporated to damp salts, and then dissolved in 1% 10 mL of hydrochloric acid solution. Ashes dissolved completely, therefore the solution did not filter. Samples were brought up to an appropriate volume, and spectrophotometrically compared against a set of standards.

Evaluation of the urine excretion rate of amine nitrogen [72]

Statistical analysis

Results

Digoxin- and furosemide-induced hypokalemia and magnesium loss

Loop diuretic furosemide caused a substantial loss of Mg both in the plasma and erythrocyte intracellular space by 30.4 ± 1.3% (0.65 ± 0.01 vs. 0.94 ± 0.04 mmol/L, p < 0.001) and 41.0 ± 1.1% (1.22 ± 0.03 vs. 2.06 ± 0.04 mmol/L, p < 0.001) (( figure 5 ); table 4( Table 4 )) compared with intact controls, respectively. Chronic usage of furosemide did not change the K level in erythrocytes compared with intact controls. The heart Mg and K levels both in furosemide treated rats and intact control rats did not differ significantly, and these data fluctuations did not fall outside the natural physiological norms. So heart Mg and K levels were 188.69 ± 9.90 μg/g and 1.756 ± 0.145 mg/g wet weight in control animals, and – 216.06 ± 8.58 μg/g and 1.639 ± 0.104 mg/g in the group of animals receiving furosemide, respectively.
Table 3 Erythrocyte Mg deficiency correction rate in digoxin treated rats after intravenously administered K,MgL-, D- and DL-aspartate stereoisomers

The effect of K,Mg L-, D- and DL-aspartates on digoxin-induced hypokalemia and magnesium loss

Both plasma Mg content and erythrocyte K content (( figure 3 )) differences between the groups receiving K,Mg L-, D- and DL- aspartates for 3, 6 and 24 hour time points were insignificant.

After 24 hrs of single K,Mg L-aspartate administration, heart K content was restored to control parameters and was significantly higher than digoxin controls by an average of 33.4 ± 11.5% (1.598 ± 0.137 vs. 1.198 ± 0.109 mg/g wet weight, p < 0.05) (( figure 4 )). In the groups of animals receiving K,Mg D- and DL-aspartates, the content of K in the heart did not change and remained below control parameters by an average of 48.2 ± 6.7% and 46.7 ± 4.1%, accordingly (0.934 ± 0.072 and 1.092 ± 0.118 mg/g wet weight, p < 0.05). Full restoration K in the heart of the rats receiving K,Mg DL- and D-aspartates was observed after one week of injections. Thus, in the group of animals receiving K,Mg D-aspartate, the K level in the heart was lower by an average of 13.1 ± 3.8%, and for the animals receiving K,Mg L- and DL-aspartates, higher by an average of 27.9 ± 8.9% (p < 0.05) and 10.3 ± 3.3% compared with intact control. In comparison with the group of animals receiving only digoxin, in the groups of animals receiving K,Mg L-, D-and DL-aspartates, heart K contents were higher by an average of 109.2 ± 14.5% (p < 0.05), 42.0 ± 6.2% (insignificant) and 80.4 ± 5.3% (p < 0.05), respectively (( figure 4 )).

The effect of K,Mg L-, D- and DL-aspartates on furosemide-induced hypokalemia and magnesium loss

Plasma Mg content differences between groups receiving K,Mg L-, D- and DL-aspartates for 3, 6 and 24 hour time points were insignificant.

So, it was shown that K,Mg L-aspartate administration leads to higher compensation of K and Mg deficiency in rats with furosemide and digoxin induced K and Mg depletion as compared with D- and DL-stereoisomers. According to the K and Mg deficiency correction rate, K,Mg aspartates may be ranged in the following order: K,Mg L-aspartate > K,Mg DL-aspartate > K,Mg D-aspartate. No toxic effects were observed when K,Mg L-, D- and DL-aspartates were administered to rats in doses of 100 mg/kg. In these studies, the physiologic changes produced by toxic serum concentrations of digoxin and furosemide (electrolytic and heart rhythm disturbances) were ameliorated in one week by the administration of K,Mg L-, D- and DL-aspartates.

Daily urine excretion of amine nitrogen and magnesium after intravenous administration of K,Mg L-, D- and DL-aspartates

Urine Mg excretion after administration of K,Mg L-aspartate did not differ from the control group in all time periods. After 4 hrs of single administered K,Mg D-and DL-aspartate urine Mg was significantly increased compared with control by an averege of 60.0 ± 9.5% and 54.1 ± 13.0% ( (figure 7) ). Urine Mg level, after 4 hrs of single K,Mg L-aspartate administration, was also significantly lower than after administration of K,Mg D-aspartate. In the other time periods, urine Mg excretion after administration of K,Mg D- and DL-aspartates did not differ significantly compared with control and K,Mg L-aspartate.

Discussion

K content in the heart was not different from the furosemide group either, and our results coincide with the study of Borchgrevink et al. [75, 76], that high doses of furosemide administered for 4 weeks did not cause any reduction of Mg and K in the myocardium. The heart K content was reduced in the rats receiving digoxin, which is associated with inactivation by digoxin the sarcolemma Na⁺-K⁺-ATPase pump.

Digitoxicity was also associated with depleted intraerythrocytic Mg concentration (1.36 ± 0.046 vs. 2.06 ± 0.046 mmol/L, p < 0.05) and reduced intraerythrocytic K concentration (26.86 ± 6.405 vs. 63.30 ± 3.726 mmol/L, p < 0.05) compared to non-toxic rats. Mean plasma Mg concentrations in the two groups were 1.10 ± 0.027 and 0.71 ± 0.034 mmol/L, respectively (p < 0.05).

Chronic usage of furosemide did not change K level in erythrocytes, but caused a substantial loss of Mg both in the plasma and erythrocyte intracellular space on the average 30.4 ± 1.3% (0.65 ± 0.01 vs. 0.94 ± 0.04 mmol/L, p < 0.05) and 41.0 ± 1.1% (1.22 ± 0.03 vs. 2.06 ± 0.04 mmol/L, p < 0.05) compared with intact controls, respectively. Our results coincide studies of Greger [77] and Quamme [78], that furosemide diminishes salt absorption in the thick ascending limb by virtue of their action on electroneutral Na⁺-K⁺-2Cl^– cotransport across the luminal membrane.

It has been shown that K,Mg L-aspartate administration leads to higher compensation of K and Mg deficiency in rats with furosemide and digoxin induced K and Mg depletion, as compared with D- and DL-stereoisomers. According to the K and Mg deficiency correction rate K,Mg aspartates may be ranged in the following order: K,Mg L-aspartate > K,Mg DL-aspartate > K,Mg D-aspartate. It was revealed that after administration of K,Mg L-aspartate, daily urine excretion of amine nitrogen and Mg is less than after D- and DL-stereoisomer administration. According to the quantity of excreted amine nitrogen and Mg in urine, K,Mg aspartates may be ranged in the following order: K,Mg D-aspartate ≥ K,Mg DL-aspartate > K,Mg L-aspartate.

It seems to be an established fact that the metabolism and utilisation of D- and L-aspartic acids differ. It has been believed for a long time that most amino acids are bioavailable in their L-isomeric form, because L-isomers are present in living systems almost exclusively [57]. It is well known that L-aspartic acid is mainly absorbed by the small intestine using a specific amino acid transporter located at the brush border membrane [58]. L-aspartic acid occupies an important position in the TCA cycle, urea cycle and nucleic acid formation pathway. By deamination or transamination, it is converted to oxaloacetic acid, an important component of the TCA cycle, and by further decarboxylation, is converted to pyruvic acid in the glycolytic pathway. In the urea cycle, by binding with citrulline, it is converted to arginosuccinic acid, and L-arginine is formed by addition of ammonia, and is itself converted to fumaric acid. It also has an important function in ammonia detoxification for L-aspartic acid is converted to L-asparagine by binding with ammonia. Carbamyl-L-aspartic acid, formed by combining L-aspartic acid and carbamyl phosphate, is a starting material for pyrimidine biosynthesis and plays an important role in purine biosynthesis as well.

D-aspartic acid is as easily absorbed as L-amino acid at the small intestinal site using a sodium ion-dependent transport system on the brush border membrane of the intestinal epithelial cells, with greater absorption taking place in the younger subject (maximum at 8 weeks old) [58]. D-amino acids in the body are thought to be metabolized into α-oxoacids by D-amino acid oxidase or D-aspartic acid oxidase [59, 60]. The oxoacids are further catalysed and utilized in mammals. So, the level of free D-amino acids in mammals’ bodies can be regulated by D-amino acid oxidase. Nevertheless, although most of the D-amino acids were oxidized by the liver, kidney and other tissues, it is possible for D-aspartic acid to pass through these enzymatic barriers and to be distributed in various tissues throughout the mammalian body [58].

In our study urine amine nitrogen after 4 hours of single K,Mg D- and DL-aspartate administration was significantly higher (by 20 times) than after administration of K,Mg L-aspartate. It is possible that part of the amine nitrogen in the urine would originate from the exogenously administered D-aspartate. In our study, the urine amino acid profile was additionally studied by ascending paper chromatography (data not presented). The Rf value of aspartic acid in the butanol-acetic acid-water (40:15:5) solvent was 0.22. It was shown that aspartic acid had higher concentration in the urine of K,Mg D- and DL-aspartate treated rats compared to K,Mg L-aspartate treated rats as it appeared in the most concentrated spots on the paper. However, in order to clarify this, the quantitative changes of D-aspartate as well as L-aspartate in urine between before and after administration is necessary.

K,Mg D-aspartate injection increases the urinary excretion of Mg. Although the cations associated with L- and D-aspartate interact independently with various substances in the body and participate in various physiological processes, complexes of K and Mg with L-stereoisomeric aspartic acid as an endogenous compound probably reveal a higher rate of transformation and utilization in organisms than K and Mg with D-stereoisomeric ones. It might be caused by differences in cellular penetration, membrane effects, and interactions with some kind of the exchangers. Generally, the role of anions and their stereoisomerity (e.g. aspartate-ion) in Mg²⁺ and K⁺ transport deserves more attention. Because of a lack of modern studies, additional pharmacological, molecular and cellular biology research is certainly necessary to confirm or refutes statements about the advantages of K,Mg L-aspartate over K,Mg DL- and D-aspartates.

The selection of a particular magnesium salt among others should take into account reliable pharmacological and toxicological data, it is necessary to determine the therapeutic ratio of the various salts before pharmacological use [69]. In our previous comparative study [71] it was found that intravenously administered K,Mg L-aspartate was more effective and less toxic than K,Mg D- and DL-aspartate. In that study the antiarrhythmic action of K,Mg L-, D- and DL- aspartate stereoisomers was assessed using the calcium chloride- and aconitine-induced arrhythmia models in rats and strophanthin K-induced arrhythmia model in guinea pigs. K,Mg L-aspartate decreased the incidence of arrhythmias to a greater extent, increased the time to onset of the first arrhythmia, decreased the percentage of rats that died and increased the duration of life after onset of the first arrhythmia in rats with arrhythmias induced by strophanthin K and calcium chloride, as compared with K,Mg D-aspartate and K,Mg DL-aspartate [71]. K,Mg L-aspartate also surpassed K,Mg D-aspartate and K,Mg DL-aspartate in parameters of acute toxicity (LD₅₀), effective dose (ED₅₀) and antiarrhythmic (therapeutic) ratio (LD₅₀/ED₅₀) in rats with aconitine-induced arrhythmia model [71](table 1).

Conclusions

Acknowledgements

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