Ion Homeostasis in the Leech: Contribution of Organic Anions

In the blood of the leech, Hirudo medicinalis, Cl⁻ balances only 30% of the cations (Boroffka, 1968; Zerbst-Boroffka, 1970). Quantitatively important anions are Krebs cycle intermediates and lactate (Zerbst-Boroffka, 1970; Zebe et al. 1981; Hildebrandt & Oeschger, 1987). However, the reported composition of the organic acid fraction and the individual concentrations of the acids vary considerably.

The present study investigates whether the observed variations are caused by different analytical procedures or physiological conditions of the animals and whether the determined organic acids represent the entire non-Cl⁻ anion equivalent needed for electroneutrality. On the basis of the results, the contribution of organic acids to ion homeostasis was investigated, in particular whether changes in inorganic ion concentrations observed after feeding on Cl⁻-rich, hypertonic blood (Zerbst-Boroffka, 1973; Wenning et al. 1980) lead to adjustments of organic acid levels.

Instead of using pooled material, as in all earlier studies, we analysed blood from individual animals using HPLC. Some of the results have appeared in abstract form (Wenning & Hoeger, 1987).

Leeches (Hirudo medicinalis) were obtained through a commercial supplier (E. Nell, Blutegelimport, Recklinghausen) and kept in open containers with 0·51 of Lake Constance water, changed weekly, containing (in mmoll⁻¹): KC1, 0·036; NaCl, 0·18; CaCl₂, 1·24; MgSO₄, 0·308 (Limnologisches Institut, Universität Konstanz). Ambient temperature was 16°C.

Leeches were kept in groups with access to air (‘non-aerated’) or with constant aeration (‘aerated’). ‘Cooled’ leeches were submerged in iced water for 10min prior to blood sampling, which was carried out on a chilled dish. Sampling of ‘noncooled’ leeches was done at room temperature.

For aerobic exercise, aerated leeches were forced to swim continuously for 15 min. They were cooled for blood sampling.

The effects of hypertonic, Cl⁻-rich meals were tested in two animal groups. Aerated leeches fed voluntarily on fresh rabbit blood (30–35 °C) or saline (in mmol l⁻¹: NaCl, 145; KC1, 5; L-arginine, 1; pH6·8; Elliott, 1986) enclosed in sheep small intestine. Within 30 min they ingested 2–5 times their body weight. Blood samples were taken 45–60 min after termination of feeding (animals cooled). For crop-loading experiments, non-aerated animals were pinned in a wax dish and kept wet. Saline (in mmol l⁻¹: NaCl, 145; KC1, 5; pH7·4) was infused via a flame-polished glass tube inserted into the crop (Zerbst-Boroffka, 1973). Blood was taken before and 1 h after crop loading from the same animal, which remained pinned and was not cooled. To detect regurgitation, Lissamine Green (SF, Chroma Gesellschaft) was added to all salines.

Leeches were pinned in a wax dish and kept wet. Blood (10–20 μl) was drawn from the dorsal vessel with glass capillaries (Zerbst-Boroffka, 1970). Operation and sampling were completed in 8–10 min. Haemocytes were removed by centrifugation (6000g, 2min; Hildebrandt, 1988). The supernatant was weighed and kept at −19°C until analysis. Crop fluid, removed with a flame-polished glass tube inserted through the pharynx into the crop, was frozen immediately.

Samples (diluted 10-fold with distilled water) were purified on an anion-exchange column (SAX, CL form, bed volume 100 μl) (Daish & Leonard, 1985). The organic acid fraction was eluted with 250 mmol l⁻¹ H₂SO₄ and analysed by HPLC using 50-100 μl of the eluate. Organic acids were separated on a C18 reverse-phase column using 6 mmol l⁻¹ phosphate buffer (pH2·45) as the mobile phase (flow rate: 0·7 ml min⁻¹). Peaks were detected at 210 nm and, in some cases, also at 254 and 280nm to exclude interfering compounds. The concentration of each acid Was calculated either from the area (malate, lactate, fumarate, α-ketoglutarate) or the height (succinate, citrate) of the peak. Values were corrected for the recovery from the extraction column determined with external standards: (% ± S.D.; N = 4–5) 94 ± 3·5 (malate), 100 ± 4·7 (lactate), 98 ± 1·3 (α-ketoglutarate), 99 ± 1·6 (citrate), 97 ± 5·1 (succinate) and 104 ± 3 (fumarate). The methodical error was less than 6 %. Pyruvate was not determined since it eluted with the solvent front. Samples were processed and analysed within 48 h. To test the peak purity of malate, samples of the corresponding fraction were rechromatographed either on a reverse-phase or on an ion-exchange column and subjected to enzymatic analysis. Samples of the H₂SO₄ extracts were used to determine concentrations of inorganic phosphate, Pi (McKee & Fyfe, 1985).

For comparison, perchloric acid extracts of blood samples were prepared as described by Zebe et al. (1981) and analysed by HPLC.

Except for special purity H₂SO₄ and H₃PO₄ (Suprapur, Merck), all chemicals were reagent grade quality.

Oxygen in the water was measured with a Clarke-type electrode (Radiometer, Copenhagen, Denmark). In aerated containers, was 20kPa. It dropped, without aeration, to a stable value: at 16°C to 5-3 ± 0·67kPa (N = 5) and at 6°C to 6·4kPa within 48h (5–13 animals).

For statistics, an unpaired, two-tailed tx-test (pooled variances) was applied using SYSTAT on a personal computer.

The terms ‘blood malate’ (lactate, Cl⁻ etc.) are often used instead of ‘malate (lactate, Cl⁻ etc.) concentration of the blood’.

For immobilization, leeches are usually pinned through their suckers in a stretched position. The effects of the imposed stress (handling, prolonged restraint) on blood organic acids were examined by comparing cooled and noncooled leeches (see Materials and methods). Cooling minimized muscular activity and mucus production, indicators of stress (Lent, 1973).

Duration of restraint had the greatest effect on blood lactate (Fig. 1). In uncooled leeches, the increase in lactate was five times that of cooled leeches (control) within the 10 min required for blood sampling and 13 times in 60 min of restraint. In contrast, it did not change in aerated and cooled animals after 15 min of muscular work (swimming; Table 1). A smaller increase in blood lactate was observed after feeding, probably owing to prolonged pharyngeal and body muscle peristalsis (Table 1). With prolonged restraint, concentrations of malate and fumarate decreased, while succinate levels increased (Fig. 1).

Inorganic ion concentrations have been determined previously in leeches (non-aerated, non-cooled) under prolonged restraint (Zerbst-Boroffka, 1970; Wenning et al. 1980). Blood [Cl⁻] was 36 ± 6mequivl⁻¹ and blood cation concentration was 131 ± 13mequivl⁻¹ (Na⁺+K⁺). Under those conditions, total measured organic acid concentrations were between 70 and 95mequivl⁻¹ and thus represented almost all non-Cl⁻ anions (Fig. 1).

Malate, whose concentration in leech blood was first measured by Zebe et al. (1981), was the most abundant anion (Table 1). In some physiological states, it accounted for half the total anion equivalents, thereby exceeding [Cl⁻]. In blood samples treated with perchloric acid, as done previously (Zebe et al. 1981), or purified on the anion-exchange column, we detected blood malate levels 2-3 times higher than previously reported (Zebe et al. 1981: 9·39mmoll⁻¹; Hildebrandt & Oeschger, 1987 : 7–13 mmol l⁻¹). The same high malate concentrations were obtained by analysing the malate fraction enzymatically.

Assuming a pH of 7·4, Pi accounted for 3 mequivl⁻¹. Citrate concentration was less than 1 mmol l⁻¹ (see also Zebe et al. 1981; Hildebrandt & Oeschger, 1987). No significant amounts of isocitrate, oxaloacetate or acetoacetate were found.

Anoxia leads to changes in energy metabolism and affects organic acid concentrations in leech tissue (Zebe et al. 1981) and blood (Hildebrandt & Oeschger, 1987). In natural habitats, ponds or lakes, long-lasting changes in and temperature occur (Hoffmann et al. 1986).

Variations in affected organic blood ions more than variations in ambient temperature (Table 1). The concentration of succinate was higher and that of α-ketoglutarate was lower in non-aerated than in aerated leeches. Malate level decreased significantly only at 6°C in non-aerated leeches. Succinate concentration increased at 25°C even in aerated leeches. Total measured acid level varied between 60 (non-aerated, 6°C) and 77 (aerated, 6° and 16°C) mequivl⁻¹ (Table 1).

At 5·3 kPa (16°C), the pH of the water dropped from 8·1 to 7·6.

To investigate whether the dramatic increase of blood [Cl⁻] after feeding (from 36 to 95mequivl⁻¹) and the increase of blood volume due to fluid resorption (Wenning et al. 1980; Hildebrandt & Zerbst-Boroffka, 1988) would lead to a reduction in.blood organic anion concentrations, results were compared after feeding on blood or on saline of similar ion composition. In both cases, succinate concentrations decreased (Table 1). Fumarate and a-ketoglutarate levels depended on the diet: both decreased after feeding on saline and α-ketoglutarate concentration increased after a blood meal.

Salt and water excretion by the nephridia have been investigated previously by measuring the time course of ionic and osmotic concentration changes in crop fluid, blood, primary urine and final urine after loading the crop with saline. The animals remained pinned while nephridial output was continuously measured by catheterization (Wenning et al. 1980; Zerbst-Boroffka et al. 1982). In this study, blood was analysed prior to and 1 h after crop-loading experiments, for comparison (data not shown). Levels of fumarate and malate decreased but, interestingly, lactate and succinate did not increase to the same extent as in the respective control group (60 min of restraint).

After feeding as well as after crop loading, total organic anion equivalents did not change significantly.

With respect to inorganic ions, the crop fluid is iso-osmotic and iso-ionic to leech blood (Zerbst-Boroffka, 1973; Wenning et al. 1980). However, animals lh after feeding on saline and those starved for 7 months had only traces of organic acids in the crop fluid. The nature of other anions in the crop (e.g. amino acids) was not investigated.

The high concentrations of organic acids in the blood of Hirudo medicinalis and the sensitivity of the HPLC method applied here allowed analysis of individual blood samples. As indicated by the low blood lactate level (Fig. 1, Table 1), immobilization by cooling and a short operation time yielded consistent organic acid levels minimally affected by the sampling procedure. Malate concentration was considerably higher than reported previously (Zebe et al. 1981; Hildebrandt & Oeschger, 1987). Differences in blood malate levels apparently result from the physiological state of the animal and the blood sampling procedure rather than from the analytical method used: the level is 40% lower in non-aerated, noncooled leeches (17·1 mmol l⁻¹, Fig. 1) than in aerated, cooled leeches (27·7mmoll⁻¹, Table 1). The values of 14mmoll⁻¹ lactate and 15 mmol l⁻¹ succinate (non-aerated, non-cooled leeches) given by Zerbst-Boroffka (1970) are consistent with our data (Fig. 1).

Considering individual variation, organic acids accounted for most of the anion ‘deficit’ between 131 ± 13 mequivl⁻¹ inorganic cations (Na⁺, K⁺) and 36 ± 6 mequivl⁻¹ inorganic anions (Cl⁻) (Wenning et al. 1980, Table 1).

Organic anions in body fluids have rarely been found in such large quantities as those reported here (70 % of total anions). In some insect larvae, they account for up to 50% of total anions (Jeuniaux, 1971). Among other oligochaetes, some Lumbricidae and the gnathobdellid leech Macrobdella decora also have low blood [Cl⁻] relative to cation concentrations (Oglesby, 1978; Wenning, 1987). It is not yet known whether organic acids serve as anions in these species.

As leeches live on a high-protein diet, organic acids derive from amino acid catabolism. After a meal, a rapid increase of NH₃ excretion is observed (Zebe et al. 1986). Transamination and deamination of amino acids feed the organic acid pool via a-ketoglutarate, as shown by its significant increase in leeches fed on blood compared to those fed on saline (Table 1). As leech blood contains negligible numbers of haemocytes (<1%; Hildebrandt, 1988), organic acids are produced in other tissues. Interestingly, the crop epithelium shows an abundance of succinate dehydrogenase activity after histochemical localization (M. Wade-puhl, U. Hoeger & A. Wenning, unpublished results).

Malate, a-ketoglutarate and fumarate concentrations are higher in the blood (this study) than in leech tissue (Zebe et al. 1981). The use of malate, the most abundant organic anion in tissue and blood, for energy storage under hypoxic conditions is obvious (Zebe et al. 1981; Zebe & Schottler, 1986), but does not explain accumulation in the extracellular fluid at normal values. Furthermore, whether the same concentration gradients between tissue and blood are maintained or whether they change according to the physiological situation is unknown.

Salt and water balance in the leech is maintained by the nephridia (Zerbst-Boroffka, 1973). After feeding, the transcompartmental flow from crop to final urine increases. This is accompanied by a transient increase of blood [Na⁺] and [K⁺] (+ 10mequivl⁻¹) and, to a much greater extent, a rise in blood [Cl⁻] (+ 50mequivl⁻¹) (Wenning et al. 1980). The latter seems to be a signal to increase nephridial salt output, since extracellular [Cl⁻] is continuously monitored by a set of receptor neurones (Wenning, 1989). Since the nephridia excrete Na⁺ and Cl⁻ in equal amounts and no organic acids (Zerbst-Boroffka et al. 1982), an increase of blood [Cl⁻] after feeding should be compensated by a decrease of organic anion levels. However, their concentrations did not change (Table 1) and, taking into account a blood volume increase of 25 % after feeding (Hildebrandt & Zerbst-Boroffka, 1988), could even increase. Thus, organic ions do not buffer transient inorganic ion changes. After ingestion of hypertonic, Cl⁻-rich saline, total anion levels (≈161 mequivl⁻¹) exceeded total cation levels (≈141mequivl⁻¹) in the blood. The moderate increase of blood osmolality (from 190 to 261 mosmolkg⁻¹ H₂O; Wenning et al. 1980) excludes additional univalent ions as a factor maintaining electroneutrality and suggests polyvalent cations.

In conclusion, Krebs cycle intermediates are the main anions in leech blood. Their individual concentrations change according to the metabolic state. To maintain electroneutrality, inorganic and organic ion homeostasis should be tightly coupled. After feeding, however, they are not down-regulated as would be expected from the dramatic increase of blood [Cl⁻]. The mechanisms maintaining blood organic acid levels clearly function independently of the mechanisms readjusting inorganic ion levels.

We thank R. Oeschger for a stimulating discussion on analysing organic acids, M. A. Cahill for correcting the English text and Drs I. Zerbst-Boroffka, E. Florey and H. O. Pörtner for helpful comments. Dr M. Wadepuhl kindly let us use some of his histochemical preparations. Supported by the Deutsche Forschungsgemein-schaft (We 745/2 and SFB 156).