Mitochondrial ATPase in the gills of the shore crabCarcinus maenas

Posterior gills (No. 7 and 8) of shore crabsCarcinus maenas were homogenized and fractionated by means of differential and density gradient centrifugation. Employment of marker enzymes Na-K-ATPase and carbonic anhydrase for plasma membranes and cytochrome oxidase for mitochondria showed that these structural elements were separated. Ultramicroscopic investigations of combined fractions confirmed the presence of the respective mitochondrial and vesicular plasma membrane structures. An ATPase which did not depend on the presence of sodium (20 mM) ions in the incubation medium but on the presence of potassium (20 mM) ions only was found in the mitochondrial fractions. The mitochondrial ATPase was tightly bound to cellular particulates and activated approximately threefold by bicarbonate (20 mM) ions. The activity of this ATPase was nearly completely inhibited by oligomycin (1 μg ml⁻¹) and greatly inhibited by low levels (5 mM) of thiocyanate and calcium ions, the K_i for Ca²⁺ being ca 4 mM. The results obtained confirm literature data on high mitochondrial densities in crab gills and allow the assumption of significant rates of energy metabolism in these organs. Considering its properties the mitochondrial ATPase is clearly distinct from crab gill Na-K-ATPase and can be measured specifically in samples containing Na-K-ATPase. Mitochondrial ATPase is therefore considered a suitable and reliable marker enzyme for mitochondria.     

Regulation of pH in the isolated perfused gills of the shore crabCarcinus maenas

Isolated posterior gills (no. 7) of shore crabsCarcinus maenas acclimated to brackish water of a salinity of 10 S were bathed and perfused with 50% sea water (200 mmol·l^-1 Na⁺), and the internal perfusate collected during subsequent periods of 5 min. During a single passage through the gill the pH of the perfusion medium decreased from ca. 8.1 to ca. 7.7, a result implying that the gill possesses structures which recognize unphysiologically high pH values in the haemolymph and regulates them down to physiological values of ca. 7.7. The calculated apparent proton fluxes from the epithelial cells into the haemolymph space amounted to 17.9 mol·g fw^-1·h^-1, a value of only 3.8% of net Na⁺ fluxes observed under comparable conditions. When 0.1 mmol·l^-1 KCN, an inhibitor of mitochondrial cytochrome oxidase, or 5 mmol·l^-1 ouabain, a specific inhibitor of Na⁺/K⁺-ATPase were applied in the internal perfusate, down-regulation of pH was no longer observed and the gill was completely depolarized, i.e. transepithelial potential differences dropped from-7.8 to 0 mV (haemolymph space negative to bath). Regulation of pH was completely inhibited by antagonists of carbonic anhydrase (0.1 mmol·l^-1 acetazolamide or 0.01 mmol·l^-1 ethoxyzolamide) applied in the perfusate. Inhibitors of Na⁺/H⁺ exchange, 0.1 mmol·l^-1 amiloride applied in the external bathing medium or in the internal perfusate, and symmetrical 0.01 mmol·l^-1 5-(N-ethyl-N-isopropyl)amiloride, as well as inhibitors of Cl^-/HCO₃ ^- exchange and Na⁺/HCO₃ ^- cotransport, 0.5 mmol·l^-1 4,4-diisothiocyanatostilbene-2,2-disulphonate or 0.3 mmol·l^-1 4-acetamido-4-isothiocyanatostilbene 2,2-disulphonate applied on both sides of the gill, and inhibitors of H⁺-ATPase, 0.05 mmol·l^-1 N-ethylmaleimide and 0.1 mmol·l^-1 N,N-dicyclohexylcarbodiimide —applied on both sides of the gill — did not alter the acidification of the perfusate observed in controls. Using artificial salines buffered to pH 8.1 with 0.75 mmol·l^-1 tris (hydroxymethyl) aminomethane instead of 2 mmol·l^-1 HCO₃ ^-, apparent proton fluxes were reduced to 11% of controls, a result suggesting that pH regulation by crab gills needs the presence of HCO₃ ^-. The findings obtained suggest that pH regulation by crab gills depends on the oxidative metabolism of the intact branchial epithelium and that carbonic anhydrase plays a central role in this process. Na⁺/H⁺ exchange, anion exchange or cotransport and active proton secretion seem not to be involved. While unimpaired active ion uptake is a prerequisite for pH regulation, ion transport itself is independent of it.Abbreviations acetazolamide (N-[sulphamoyl-1, 3, 4-thiadiazol-2-yl]-acetamide) - amiloride 3,5-diamino-6-chloropyrazinoyl-guanidine - CA carbonic anhydrase - DBI dextrane-bound inhibitor thiadiazolesulphonamide - DCCD N N dicyclohexylcarbodiimide - DIDS 4,4-diisothiocyanato-stilbene-2,2-disulphonate - EIPA 5-(N-ethyl-N-isopropyl) amiloride - ethoxyzolamide 6-ethoxy-2-benzothiazole-sulphonamide - fw fresh weight - J _H ⁺ apparent proton flux - NEM N-ethylmaleimide - PD transepithelial potential difference - PEG-STZ polyethylene-glycol-thiadiazolesulphonamide - STTS 4-acetamido-4-isothiocyanatostibene 2,2-disulphonate - SW sea water - TRIS tris(hydroxymethyl)aminomethane     

Active uptake of sodium in the gills of the hyperregulating shore crabCarcinus maenas

Isolated posterior gills of shore crabs,Carcinus maenas, previously acclimated for at least 1 month to brackish water of 10 S, were connected with an artificial hemolymph circulation by means of thin polyethylene tubings. Gills were symmetrically perfused and bathed with 50 % sea water. Transepithelial potential differences (PDs) and fluxes of sodium between medium and blood were measured under control conditions and following reductions of PDs by means of 5 mM internal (blood side) ouabain, 0.5 mM internal and external (bathing medium) NaCN or by exhaustion of energy reserves along with a prolonged perfusion period of more than 9 h. In these experiments²²Na was used as tracer. Each of the three modes of reducing transepithelial potential differences resulted in a decrease in sodium influxes from 500–1000 µmoles g^–1 h^–1 to 250–400 µmoles g^–1 h^–1. The findings suggest that sodium influx, which normally greatly exceeds efflux, was diminished by its active component. The remaining non-inhibitable influx equals efflux values. Our findings thus indicate that efflux is completely passive, while influx has — beside a passive component of efflux magnitudes — an additional active portion which is much larger than the passive component. Since ouabain is a specific inhibitor of the Na-K-ATPase, our results confirm previous findings (Siebers et al., 1985) that the basolaterally located Na-K-ATPase generates the transepithelial potential difference in the gills, which is inside negative by about 6–12 mV. Inhibition of the active portion of sodium influx by internal ouabain along with reduced PDs suggests that transepithelial PDs generated by the branchial sodium pump are the driving force for active sodium uptake in hyperregulating brackish water crabs.     

Structural elements of the gills of the shore crabCarcinus mediterraneus

Fine structural studies were conducted on the gills of the shore crabCarcinus mediterraneus using scanning electron microscopic techniques. The results obtained show the structural organization of crab gills from whole gills including spiny elements over the 150 lamellae to lamellar components such as cuticles, median shaft, marginal canal, afferent and efferent lamellar vessels and hemolymph cells. Enormous surface enlargement is accomplished by a variety of structural elements which allow rapid circulation of hemolymph. In the form of a relatively small organ, the gills fulfill all the necessary exchanges of specific molecules between the crab and its environment. Aggregations of ca 1-μm particles covering the outer cuticular surfaces are considered to be bacterial colonies of unknown properties and functions.     

The role of the transbranchial potential difference in hyperosmotic regulation of the shore crabCarcinus maenas

When isolated gills of the shore crabCarcinus maenas were bathed and perfused with identical solutions on both sides (50 % sea water), a spontaneous transepithelial potential difference (PD) of some millivolts (hemolymph side negative) was established. This PD is of active nature and requires the metabolism of the living cell, since it uses its own sources of energy in addition to organic nutrients offered in the flow of artificial hemolymph. Addition of sodium cyanide and dinitrophenole to bathing and perfusion medium resulted in reversible breakdown of PDs in a concentration-dependent mode. In posterior gills ofC. maenas, the potential differences were more negative compared to data measured in anterior gills of the same individuals. These results are correlated with higher specific activities of Na-K-ATPase in posterior gills. Experiments with triamterene indicate that sodium uptake inC. maenas is sensitive to this diuretic drug, when applied on the apical side of the epithelial cell. The results obtained show that active uptake of sodium from medium to blood across the gills is performed by a complex mechanism including participation of several basal and apical transport steps.     

Regulation of sodium in the shore crabCarcinus maenas,adapted to environments of constant and changing salinities

The activity of Na-K-ATPase was determined in the posterior gills of the shore crabCarcinus maenas during a period following transfer from 35 to 10 ‰ salinity and vice versa at 15 °C. After transfer from high to low salinity, Na-K-ATPase activity increased from 3.2 to 7.0 μmoles P_i mg protein^?1 h^?1 within a period of 2 to 3 weeks. Transfer of crabs from low to high salinity resulted in reduction of activity from 7.4 to 4.5 μmoles P_i mg protein^?1 h^?1 within about the same period. The relatively slow response following salinity change indicates that the amounts of Na-K-ATPase in the gills may play a role in hyperionic Na regulation in relatively constant brackish-water environments. Instant responses to salinity result from activation and inhibition of Na-K-ATPase activity by Na. Gill Na-K-ATPase is activated by the Na concentration of the incubation medium to attain a steep maximum at about 75 mM Na, which corresponds to the lowest environmental Na levels tolerated byC. maenas equivalent to a salinity of ca 6 ‰. Activity greatly decreased towards higher Na levels, equivalent to the salinity of normal sea water, at which hyperregulation no longer occurs. Selective addition of either Na or Cl to brackish water of 9 ‰ S resulted in effective hyperregulation of the non-increased ion, and passive distribution between medium and blood of the increased ion. These data indicate that under appropriate conditions the normally coupled transport of Na and Cl may be uncoupled and take place independently of each other.     

A membrane-bound hemoglobin from gills of the green shore crab Carcinus maenas

Most hemoglobins serve for the transport or storage of O(2). Although hemoglobins are widespread in "entomostracan" Crustacea, malacostracans harbor the copper-containing hemocyanin in their hemolymph. Usually, only one type of respiratory protein occurs within a single species. Here, we report the identification of a hemoglobin of the shore crab Carcinus maenas (Malacostraca, Brachyura). In contrast to the dodecameric hemocyanin of this species, C. maenas hemoglobin does not reside in the hemolymph but is restricted to the gills. Immunofluorescence studies and cell fractioning showed that C. maenas hemoglobin resides in the membrane of the chief cells of the gill. To the best of our knowledge, this is the first time that a membrane-bound hemoglobin has been identified in eukaryotes. Bioinformatic evaluation suggests that C. maenas hemoglobin is anchored in the membrane by N-myristoylation. Recombinant C. maenas hemoglobin has a hexacoordinate binding scheme at the Fe(2+) and an oxygen affinity of P(50) = 0.5 Torr. A rapid autoxidation rate precludes a function as oxygen carrier. We rather speculate that, analogous to prokaryotic membrane-globins, C. maenas hemoglobin carries out enzymatic functions to protect the lipids in cell membrane from reactive oxygen species. Sequence comparisons and phylogenetic studies suggested that the ancestral arthropod hemoglobin was most likely an N-myristoylated protein that did not have an O(2) supply function. True respiratory hemoglobins of arthropods, however, evolved independently in chironomid midges and branchiopod crustaceans.     

Salinity-mediated carbonic anhydrase induction in the gills of the euryhaline green crab, Carcinus maenas

The euryhaline green crab, Carcinus maenas, is a relatively strong osmotic and ionic regulator, being able to maintain its hemolymph osmolality as much as 300 mOsm higher than that in the medium when the crab is acclimated to low salinity. It makes the transition from osmoconformity to osmoregulation at a critical salinity of 26 ppt, and new acclimated concentrations of hemolymph osmotic and ionic constituents are reached within 12 h after transfer to low salinity. One of the central features of this transition is an 8-fold induction of the enzyme carbonic anhydrase (CA) in the gills. This induction occurs primarily in the cytoplasmic pool of CA in the posterior, ion-transporting gills, although the membrane-associated fraction of CA also shows some induction in response to low salinity. Inhibition of branchial CA activity with acetazolamide (Az) has no effect in crabs acclimated to 32 ppt but causes a depression in hemolymph osmotic and ionic concentrations in crabs acclimated to 10 ppt. The salinity-sensitive nature of the cytoplasmic CA pool and the sensitivity of hemolymph osmotic/ionic regulation to Az confirm the enzyme's role in ion transport and regulation in this species. CA induction is a result of gene activation, as evidenced by an increase in CA mRNA at 24 h after transfer to low salinity and an increase in protein-specific CA activity immediately following at 48 h post-transfer. CA gene expression appears to be under inhibitory control by an as-yet unidentified repressor substance found in the major endocrine complex of the crab, the eyestalk.     

Mechanisms of intracellular isosmotic regulation: Extracellular space of the shore crabCarcinus maenas in relation to environmental salinity

Summary 1. In shore crabsCarcinus maenas, which had been maintained for one month in 10, 20, 30, 40, and 50 salinity, the extracellular space was estimated by means of the dilution of the food dye amaranth and the polyglycan¹⁴C-inulin.2. The extracellular space does not differ significantly in response to the external salinity. It varies between 29.2 and 36.0% (with a mean of 33.1%) of body weight when estimated by dilution of amaranth and between 16.7 and 18.5% (with a mean of 17.9%) of body weight when estimated by¹⁴C-inulin dilution. Differences of values in regard to the substances used for estimation are discussed.3. The results confirm that the magnitude of the extracellular space is not involved in phenomena of osmoregulation.4. The rapid reduction in the concentration of haemolymph proteins after transfer of freshwater and marine decapods from lower to higher salinities (Drilhon-Courtois 1934,Siebers et al. 1972) implies, on the basis of the present results, also a reduction in the total amount of haemolymph protein in the crab, since the protein-distribution volume remains unchanged within a wide variety of salinities. The assumption that haemolymph proteins participate in the increase of intracellular free amino acids during isosmotic intracellular regulation is confirmed.

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Mechanismen der intrazellulären isosmotischen Regulation: Der extrazelluläre Raum der StrandkrabbeCarcinus maenas in Abhängigkeit vom Salzgehalt

Kurzfassung Durch Verdünnungsanalyse des markierten Polysaccharids¹⁴C-Inulin und des Nahrungsmittelfarbstoffes Amaranth nach Injektion ins Hämocoel wurde der extrazelluläre Raum vonCarcinus maenas bestimmt. Die Strandkrabben waren einen Monat lang in Salinitäten von 10–15 gehalten worden. Die Größe des extrazellulären Raumes, die sich als weitgehend unabhängig vom Salzgehalt erwies, betrug 29,2 bis 36,0% des Körpergewichts (mit einem Mittel von 33,1%) bei Verdünnungsanalyse von Amaranth bzw. 16,7 bis 18,5% des Körpergewichts (mit einem Mittel von 17,9%) bei Verdünnungsanalyse von¹⁴C-Inulin. Die unterschiedlichen Ergebnisse, die bei den Bestimmungen mit den beiden Substanzen erhalten worden sind, werden diskutiert. Die weitgehende Konstanz des extrazellulären Raumes trotz beträchtlicher Unterschiede im Salzgehalt zeigt, daß der extrazelluläre Raum kaum einen Einfluß auf die osmoregulatorischen Vorgänge hat. Die rasche Abnahme der Hämolymphproteinkonzentration nach Überführung verschiedener Süßwasser- und Meereskrebse (Drilhon-Courtois 1934,Siebers et al. 1972) in höhere Salinitäten stellt sich somit als eine Verminderung des gesamten Hämolymphproteins im Tier dar, da die Verteilungsräume für das Protein konstant bleiben. Es ist daher wahrscheinlich, daß nach einem Salinitätsanstieg Hämolymphproteine bei der Erhöhung der Konzentration intrazellulärer freier Aminosäuren beteiligt sind.

     

Neuroendocrine regulation of carbonic anhydrase expression in the gills of the euryhaline green crab, Carcinus maenas

The relationship between branchial carbonic anhydrase (CA) activity, CA gene expression and salinity, and potential mechanisms of regulation, was investigated in the euryhaline green crab, Carcinus maenas, acclimated to 33 ppt and transferred to 10 ppt, and the stenohaline rock crab, Cancer irroratus, acclimated to 32 ppt and transferred to 18 ppt. CA activity in green crabs acclimated to high and low salinity was a function of CA mRNA expression, with low salinity exposure resulting in an increase in both CA expression and activity. Eyestalk ablation (ESA) in green crabs acclimated to high salinity resulted in an increase in CA expression in the posterior, ion-transporting gills, in the absence of the low salinity stimulus. There were no changes in CA activity or expression in the anterior, respiratory gills. ESA also potentiated low salinity-stimulated CA induction, again, only in posterior gills. There were no changes in CA activity in any gills of Cancer irroratus, in response to either ESA or low salinity. These results suggest that CA expression in euryhaline, osmoregulating species, is under inhibitory regulation by a putative repressor found in the eyestalk, and that this mechanism is absent in stenohaline, osmoconforming species. CA expression is maintained at low, baseline levels in crabs acclimated to high salinity by the presence and action of this compound. The effects of the repressor appear to be reduced upon exposure to low salinity, allowing CA induction to occur.     

Sodium currents in the giant axon of the crabCarcinus maenas

Summary Measurements were made of the kinetics and steady-state properties of the sodium conductance changes in the giant axon of the crabCarcinus maenas. The conductance measurements were made in the presence of small concentrations of tetrodotoxin and as much electrical compensation as possible in order to minimize errors caused by the series resistance. After an initial delay of 10–150 sec, the conductance increase during depolarizing voltage clamp pulses followed the Hodgkin-Huxley kinetics. Values of the time constant for the activation of the sodium conductance lay on a bell-shaped curve with a maximum under 180 sec at –40 mV (at 18°C). Values of the time constant for the inactivation of the sodium conductance were also fitted using a bell-shaped curve with a maximum under 7 msec at –70 mV. The effects of membrane potential on the fraction of Na channels available for activation studied using double pulse protocols suggest that hyperpolarizing potentials more negative than –100 mV lock a fraction of the Na channels in a closed conformation.     

Potassium currents in the giant axon of the crabCarcinus maenas

Summary Measurements were made of the kinetic and steadystate characteristics of the potassium conductance in the giant axon of the crabCarcinus maenas. These measurements were made in the presence of tetrodotoxin, using the feedback amplifier concept introduced by Dodge and Frankenhaeuser (J. Physiol. (London) 143:76–90). The conductance increase during depolarizing voltage-clamp pulses was analyzed assuming that two separate potassium channels exist in these axons. The first potassium channel exhibited activation and fast inactivation gating which could be fitted using them ³ h, Hodgkin-Huxley formalism. The second potassium channel exhibited the standardn ⁴ Hodgkin-Huxley kinetics. These two postulated channels are blocked by internal application of caesium, tetraethylammonium and sodium ions. External application of 4 amino-pyridine also blocks these channels.     

Mechanisms of intracellular isosmotic regulation: Fate of14C-labeled serum proteins in the shore crabCarcinus maenas after changed environmental salinity

Summary 1. Two individuals of the common shore crabCarcinus maenas, which had been maintained for 2 months in 11 S, were injected 100 µCi of¹⁴C-glucose and a¹⁴C-amino acid mixture in physiological saline during 5 days. They were the source of a¹⁴C-labeled serum protein, which was injected into the hemocoel of crabs which had been maintained for more than 2 months in 11 S. Six hours after injection the distribution of label and concentrations of serum proteins and free amino acids were investigated in 13 crabs, transferred to 38 S after the injection, and in 13 crabs (controls) which remained in 11 S.2. In crabs transferred from 11 to 38 S, proteolytic processes in serum increased; this was demonstrated by a decrease in serum proteins 6 hours after transfer. This assumption is confirmed by the fact, that¹⁴C-radioactivity in serum originating from serum proteins decreased by 44.4 % within 6 hours after transfer, whereas in controls it decreased by 32.6 %.3. Serum proteins have been metabolized in favour of low molecular intracellular intermediary products, such as amino acids, sugars, and organic acids, all contributing to the new intracellular osmotic pressure. This is shown by a significant increase in radioactivity of low molecular intracellular intermediary products.

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Mechanismen der intrazellulären isosmotischen Regulation: Das Schicksal von¹⁴C-markiertem Serumprotein der StrandkrabbeCarcinus maenas nach einem Wechsel der äußeren Salinität

Kurzfassung Individuen der StrandkrabbeCarcinus maenas, die zwei Monate in 11 S gehalten worden waren, wurden innerhalb von 5 Tagen 100 µCi¹⁴C-Glucose und eines¹⁴C-Aminosäuregemisches in physiologischer Lösung ins Hämocoel injiziert. Diese Tiere dienten als Quelle der benötigten¹⁴C-Serumproteinlösung, die anderen, über 2 Monate in 11 S gehaltenen Strandkrabben ins Hämocoel injiziert wurde. Eine Gruppe dieser Tiere wurde nach der Injektion in 38 S überführt, die Kontrollgruppe verblieb nach der Injektion in 11 S. 6 Stunden später wurden die Konzentrationen von Serumproteinen und freien Aminosäuren sowie die im Serum verbliebene Radioaktivität und die Radioaktivität in den gesamten niedermolekularen Intermediärprodukten bestimmt. Aus der signifikanten Abnahme des Serumproteins und der im Serum verbliebenen Radioaktivität sowie aus einer signifikanten Zunahme der Radioaktivität in den osmotisch aktiven Intermediärprodukten wird geschlossen, daß die intrazelluläre niedermolekulare organische Substanz, die während der Osmoregulation bis zu 100 % vermehrt werden kann, zu einem erheblichen Teil durch proteolytische Vorgänge im Serum der Tiere entsteht.

     

Inhibition of carbonic anhydrase in the gills of two euryhaline crabs,Callinectes sapidus and Carcinus maenas,by heavy metals

Two subcellular fractions of gill tissue, cytoplasm and basolateral membranes, from two species of euryhaline decapod crustaceans, Callinectes sapidus and Carcinus maenas, acclimated to low salinity, were isolated via differential centrifugation. Carbonic anhydrase activity from both fractions was titrated against a variety of heavy metals in vitro. The metals Ag(+), Cd(2+), Cu(2+) and Zn(+) showed inhibitory action against the enzyme. Ki values for these metals against cytoplasmic CA from C. sapidus were in the range of 0.05-0.5 microM (for Ag(+), Cd(2+) and Cu(2+)) and 2-6 microM for Zn(+), some of the highest sensitivities reported for CA from an aquatic organism. The Ki values for these same metals were approximately 2-3 orders of magnitude higher for cytoplasmic CA from C. maenas, indicating that there are significant differences in heavy metal sensitivity in branchial CA from the two species, and that C. maenas possesses a metal-resistant CA isoform. It required concentrations of metals in the millimolar range, however, to inhibit CA activity from the membrane fraction of the gill of both species. There were no effects on either mortality or on hemolymph osmotic and ionic concentrations in C. maenas that were exposed to 10 microM Cd or Zn(+) at 32 per thousand salinity and subsequently transferred to 10 per thousand. The presence of a metal-resistant CA isoform in the gills of C. maenas suggests that this species would not be restricted from its normal estuarine environment by heavy metal pollution.     

Carbonic anhydrase activity in the blood and the gills of rainbow trout during long-term hypercapnia in hard,bicarbonate-rich freshwater

Summary Carbonic anhydrase (CA) activities in gills and venous blood, acid-base balance, and haematological variables were studied during environmental hypercapnia in rainbow trout (Salmo gairdneri). Batches of 8–10 fish were exposed to about 3 or 13 mmHg in flow-through tests of various duration from 4 h to 80 days.After initial acidosis, blood pH rose above pre-experimental values. At 3 mmHg it became normal again within 21 days, while at 13 mmHg the overshoot lasted for 80 days. In fish acclimated for 3 weeks or more to 13 mmHg , blood HCO ₃ ^– increased four to five times while plasma Cl^– levels were lower and K⁺ higher. Na⁺ levels did not show any consistent trend associated with exposure to hypercapnia. After an initial acidaemia, Hct, Hb, and RBC remained relatively constant.Patterns of change in CA activity differed between gills and erythrocytes. Initially, blood CA decreased at both levels. It then began rising after about 3 weeks and tended to reach pre-experimental values by 80 day's hypercapnia. At 13 mmHg , gill CA increased to twice the pre-experimental level. Compared with blood CA, gill CA appeared to be more specifically involved in fish acclimation to hypercapnia, which demands an increase in blood bicarbonate to provide a sufficient buffering capacity. Increased CA indicates that the gill enzyme may play a more important role than blood CA in acid-base regulation in fish during hypercapnia.Abbreviations CA carbonic anhydrase - Hb haemoglobin - Hct haematocrit value - RBC red blood cells