NW/. Med. Biol. Vol. 16, No. 6, pp. 609-61 I, 1989 IN. J. Radial. Appl. Instrum. Parr B Printed in Great Britain. All rights reserved

0883-2897189 S3.00 + 0.00 Copyright 0 1989 Pergamon Press plc

Utilization of Metal Ions Administered as Labelled Salts of Phosphatidic Acid

PAVEL RAUCH, JAN KAS and MOJMfR RANNQ

Department of Biochemistry and Microbiology, Institute of Chemical Technology, 166 28 Prague, Czechoslovakia

(Received 6 December 1988)

Phosphatidic acid is used in food technology as an emulsifier. Simultaneously, it can serve as a carrier for different biologically important metal ions. This paper concerns the stability of phosphatidic acid salts in body fluids and the incorporation and distribution of metal ions into the organs of experimental animals. It was found that the absorption of metals, released from salts of phosphatidic acid after administration, is comparable with the absorption of easily dissociable inorganic salts.

Introduction

Many authors have found that the biologically im- portant metal ions Co, Mn, Zn, Cu and Mg are better utilized from foodstuffs when they are administered in the form of organic compounds (Sinha and Prasad, 1981) or as a part of food (e.g. previously incorpor- ated into chlorella (Ichikawa et al., 1980), or fish (Inaba et al., 1982). Thus attention must be paid to the synthesis of different substances which can be used as effective carriers of metal ions. Phosphatidic acid (PA) is considered to be a suitable carrier for this purpose. Synthetic salts of PA are used widely as emulsifiers in food technology instead of lecithins isolated from natural sources (Goldfine, 1968; WHO, 1970). The metabolic fate of PA, labelled with 32P, after oral administration to rats has been elucidated recently (Rauch and K& 1984). Similarly, the role of PA as a carrier for calcium, iron alone or in mixed preparations was investigated (Rauch and KBS, 1984a; Rauch er al., 1984). PA may serve, however, as a carrier of other biological metal ions, for instance Colt, Mn2+, Zn2+, Cu2+ and Mg2+, and fulfil further functions in the organism, e.g. as a precursor of phospholipid biosynthesis (Jacobson and Papahadjopoulos, 1975).

The aim of this paper was to investigate the utilization of the above mentioned metal ions admin- istered as salts of PA of rats and to compare it with corresponding easily dissociable inorganic salts.

Materials and Methods

Materials

Synthetic phosphatidic acids containing saturated fatty acids were supplied by Milo, Olomouc,

Czechoslovakia. s8CoC12, tiCuC12, 54MnC12 and 6’ZnC12, all having a radioactivity of 370 MBq/S mL, were obtained from the Institute of Nuclear Re- search, Swierck, Poland. The tissue solubilizer Proto- sol and the liquid scintillator Aquasol were purchased from NEN Chemicals, Boston, U.S.A. All other chemicals used were obtained from Lachema, Bmo, Czechoslovakia. White, male rats weighing about 170 g were supplied from the Experimental Animal Farm at Lysolaje near Prague, Czechoslovakia.

Methods

Synthesis of acetates of metal ions. MnCl, (8 m- equiv) and “MnCl, (370 MBq) in a volume of 5 mL was precipitated with 3 M Na,CO, at pH 10. The precipitate formed was washed well with ice-cold water and carefully dried in a stream of nitrogen. The manganese carbonate was then dissolved in acetic acid and the resultant acetate dried in a stream of nitrogen at 50°C. The acetates of Yu, s8Co and 6SZn were prepared in a similar way.

Synthesis of salts of phosphatidic acid. Five grams of phosphatidic acid was added to an Erlenmeyer flask (10 mL) containing freshly prepared Mn- acetate. The temperature was raised to 80°C and the reaction was allowed to proceed with constant stir- ring for 1 h. The resulting preparation had a specific radioactivity of 50 MBq/g. The other salts of phos- phatidic acid were prepared in a similar manner.

Administration of samples. The metal salts of phos- phatidic acids were administered to rats in 100 mg doses by the technique described earlier (Rauch and KIS, 1984). Metal chlorides with same specific radio- activity were administered to the control groups of rats. The animals were kept in metabolic cages (Rauch and K6S, 1984a).

609

610 PAVEL RAUCH et al.

Determination of solubility of biogenic metal ions in gastric juice and intestinal homogenate. Phosphatidic acid salts (300mg) were added to 5 mL of human saliva and gastric juice or to 20% intestinal mucosa suspension in Tris-HCI buffer, pH 8.3. The suspen- sion was incubated at 37°C for 3 h under constant stirring. The released metal ion concentrations were determined in supernatants by the following spec- trophotometric methods: Co with L-nitroso-2-naph- tol, Cu with diethyldithiocarbamate, magnesium with oxine, Mn with hydroxylamine hydrochloride, and Zn with dithizone (Koch and Koch-Dedic, 1974).

Preparation of samples for measurement of radio- activity. Samples of tissue (50 mg) were solubilized by the procedure described elsewhere (Rauch and KG, 1984a). The radioactivity of @Cu was determined by liquid scintillation counting in a Packard-Tricarb instrument. The radioactivity of ‘*Co, 54Mn and 65Zn was determined in a well-type crystal scintillation detector using the procedure described elsewhere (Rauch et al., 1986).

Results

Breakdown of metal salts of phosphatidic acid contain - ing in body fluids in vitro

The release of metal ions from salts of phosphatidic acids in human stomach juice or in 20% intestinal mucosa is shown in Table 1. The amounts of the solubilized metal ions were determined after incu- bation in vitro at 37°C for 3 h. The values given are the arithmetic means of six parallel determinations. The amounts of metal ion released in stomach juice varied from about 12% for Co2+ up to 35% for Mg2+ of the total metal ion bound in the salt. Higher degrees of dissociation were found in the intestinal

homogenate, from 20% up to 50%, while dissoci- ation in saliva is almost negligible (Table 1).

Metal ion incorporation into rats after administration of radiolabelled salts of phosphatidic acid

The values of the incorporated radioactivity of 58Co “Mn 65Zn and “Cu into 10 selected rat tissuks are given in Table 2. The highest metal ion incorporation was found in liver and decreases in the following order: kidney > heart > spleen > lung. The radioactivity incorporation into the other organs was much lower. Simultaneously, the incorporation from the easily dissociable inorganic salts was also investigated.

The results obtained were evaluated by the Stu- dent’s t-test. “Mn incorporation from “Mn-PA was not significantly different (P > 0.3) from the incor- poration of MnCl,. In contrast , Wo, 65Zn and 64Cu incorporation from PA salts was significantly lower in comparison with the corresponding inorganic salts.

Comparison of metal ion incorporation after adminis- tration as phosphatidic acid and inorganic salts

The mean overall incorporation was calculated from the ratios given in Table 2 which represents the arithmetic means of values from ten experimental animals. The values for the mean incorporation were used for the calculation of metal ion utilization administered in the form of PA salts. The metal ion utilization from the inorganic salt was taken as 100%.

Discussion

The stability of PA salts in different parts of the gastrointestinal tract was investigated by the experiments in vitro and evaluated on the basis of their dissociation rate in saliva, stomach juice and

Table 1. Dissociation of phosphatidic acid salts, containing biologically important metal ions, in body fluids in vitro (37°C. 3 h)

Body fluid co

Amount of released metal ion (%)*

Mn Zn cu Mg

Saliva 0.5 f 0.1 0.8 + 0.2 0.7 + 0.1 0.8 f 0.2 0.9 +_ 0.1 Gastric iuice 12+2 33 + 2 25+ I 30 + I 35 + 2 Intestinal mucosa 20 : 4 40 + 4 45 I3 47 I4 5oz3

‘Values are arithmetic means of six parallel determinations & SD.

Table 2. Incorporation of metal ions into rat organs after per ore application of phosphatidic acid salts and inorganic salts (n = IO)

Incorporated radioactivity expressed in % dose administered/p. tissue f SD

Organ Co-PA

Liver 2.4 f 0.3 Kidney 1.2 f 0.2 Heart 0.6 k 0. I Spleen 0.5 f 0.1 Lung 0.3 * 0.1 Bone 0.3 + 0.1 Skin 0. I f 0.03 Muscle 0.1 i 0.03 Tooth 0.1 f 0.02 Blood ND

Co-PA

co

0.63 0.63 0.68 0.69 0.71 0.75 I .oo 0.66 0.80

Mn-PA

20.2 i 2.8 9.4 f 1.4

10.7 * I.7 6.8 f 1.7 2.8 + 0.4 I.8 50.2 0.6 + 0. I 0.7 f 0.1 I.5 io.2 0.2 f 0.03

Mn-PA Zn-PA

Mn Zn-PA Zn Q-PA

I .02 12.1 + I.1 0.75 10.3 i: I.8 0.97 1.3 + 0.7 0.73 5.7 + I.2 1.00 I.8 * 0.2 0.59 3.1 k 0.6 I.16 I.2 +0.2 0.87 I.9 + 0.3 0.94 2.2 + 0.2 1.01 I.1 kO.2’ I .07 0.8 f 0.1 0.74 0.5 f 0.1 I .oo 0.4 * 0.1 0.60 0.2 + 0.06 0.92 0.7 f 0.1 0.80 0.3 + 0.06 I .09 0.5 f 0.1 0.80 0.5 t 0.05 0.75 0.2 + 0.03 0.80 ND

Cu-PA

cu

0.85 0.73 I .23 0.86 I .06 0.73 0.75 0.83 0.76

n = number of animals. ND = nondetectable radioactivity.

Utilization of metals 611

intestinal mucosa. It was found that the dissociation of PA salts in human saliva is unimportant because it did not exceed 1% for all the salts tested. The dissociation of PA salts in stomach juice was much higher (see Table 1) and facilitated absorption of the metal ions. Even higher dissociation of PA salts occurs with the intestinal mucosa. It is possible to anticipate that under in vivo conditions the dissoci- ation of PA salts will be increased due to the emulsi- fying properties of the secreted bile acids and therefore the utilization of metal ions will also be enhanced. It may be assumed that the stepwise release of metal ions in stomach and intestine and the subsequent resorption will facilitate the dissociation of further molecules of the PA salt thus making additional metal ions available for incorporation. This idea is supported by our earlier results achieved with calcium and iron salts of PA (Rauch and KBS, 1984a; Rauch et al., 1984).

The incorporation of metal ions into rat organs after per ora administration of PA salts is greatest in liver and kidney and may be related to the metabolic activity of the tissue (Table 2). The majority of the administered radioactivity is incorporated into five important organs in decreasing order: liver > kidney > heart > spleen and lung. Tissue extracts in 5% trichloroacetic acid and lipid extracts in methanol : chloroform, 1: 1 were prepared from these organs. The results showed that the metal ions administered in the form of their PA salts are incor- porated in the same way as the dissociated ions released from the inorganic salts in the digestive tract. The labelled metal ions were found completely in the acid extracts while the lipid extracts were free from radioactivity. The maximal incorporation of the investigated metal ions into rat organs was detected in the period between 24 and 48 h following the administration.

Simultaneously, the incorporation of metal ions from the inorganic salts was investigated. Very good utilization of metal ions in the organism of the experimental animals was found (Table 2). Man- ganese is incorporated after its administration in the form of PA salts as well as in the form of inorganic salts. The other metal ions tested are incorporated significantly less as PA salts, however, their incor- poration is still quite high, e.g. 85% for copper and 75% for zinc. This lower utilization of metal ions is compensated by the positive effect of the simul- taneous supplementation with PA, which is beneficial from the view of nutrition (fortification with phos- pholipids) and technology as an emulsifier (Goldfine, 1968; WHO. 1970).

The application of the phosphatidic acid metal salts as components of animal feeds as well as to pharmaceutical preparations is expected to be very small owing to the low requirements of human and animal organisms for the metal ions investigated. The daily consumption of the metals per individual and day is calculated to be 0. l-l .O mg for Co, 3-9 mg for Mn, 2-5 mg for Cu, 5-22 mg for Zn and up to 300 mg for Mg (Underwood, 1971). The further advantage of the application of phosphatidic acid salts as carriers for metal ions is the possibility of combined prep- arations containing, besides the above mentioned metal ions, iron and calcium. The preparation of combined salts of PA offers the possibility of simul- taneous supplementation with several metal ions in nutrition or in the therapy of both human and animal deficiency diseases. Thus PA salts may be considered preferable as carriers of deficient metal ions to other types of preparations which permit the fortification with only one metal element.

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