Comp. Biochem. Physiol. Vol. 83B, No. 4, pp. 843-846, 1986
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GLUTATHIONE IN THE RED BLOOD CELLS OF EMBRYONIC MICE THOMAS BRITTAIN* and BRONWYN TOTTLE Department of Biochemistry, University of Auckland, Auckland, New Zealand (Received 12 September [985) Abstract--l. A micro-method for the determination of red blood cell glutathione levels has been
developed. 2. It has been shown that, in order to obtain true values of glutathione concentrations within red blood cells, it is necessary to correct for loss of labelled glutathione and also to take account of the time taken to complete the analytical procedure. 3. Glutathione is the major small molecular weight thiol present in embryonic red blood cells, 4. The glutathione to haemoglobin ratio is maintained at 0.6 from 13 days gestation to adulthood in the mouse. 5. Decay of glutathione in both adult and embryonic red blood cells can be avoided by incubation of the red blood cells in glucose containing buffers.
INTRODUCTION Although glutathione was discovered in 1888 by de Rey-Pailhode it was not until 1921 that Hopkins first crystalised this thiol containing tripeptide. Since that time glutathione has been shown to be present in almost all mammalian cell types in concentrations ranging from 1-10 mM (Meister, 1975). In particular it is the major small molecular weight thiol present in adult mammalian erythrocytes, being present at about half the concentration of the haemoglobin present (Meister, 1975). Over the years a wide range of studies have been reported concerning the concentration of glutathione present in the red blood cells of various mammals (Agar et al., 1974; Yu and Agar, 1979; Moron et al., 1979) and the effects of various agents on these levels (Mahaffey and Smith, 1975; Raheja et al., 1983; Srivastava et al., 1974). Invariably these studies have been carried out on the adult of the species with only mention being made of the glutathione levels in the new born (Agar et al., 1974). As the adult red blood cell in mammals is almost devoid of the normal cellular enzyme systems its response to oxidative challenge must be limited. At the embryonic stage the red blood cell is nucleated and capable of de novo protein synthesis. As a continuation of our previous studies on embryonic red blood cells (Purdie et al., 1983, Wells and Brittain, 1983; Brittain and Wells, 1983) we have investigated the levels of glutathione in these cells. In order to carry out these studies it has been necessary to develop a micro scale method to measure glutathione levels. In the past a wide range of methods have been employed for the determination of glutathione levels in cells. Commonly, labelling of glutathione with 55'-dithio-bis(2-nitrobenzoic acid) has been employed (Beutler, 1971) to yield a coloured product with an absorption at 412 nm. A fluorescent *Correspondence to be addressed to Dr T. Brittain, Department of Biochemistry, University of Auckland, Auckland, New Zealand (Tel: 737-999).
method employing reaction of glutathione with ophthalaldehyde has been used (Cohn and Lyle, 1966; Srivastava and Beutler, 1968) as well as a thiodisulphide exchange method (Davidson and Hird, 1964). These methods do not distinguish between glutathione and other thiols and rely on the assumption that glutathione is the only major thiol present. This problem has to some extent been alleviated by the use of an enzymic system which requires glutathione as a substrate (Bernt and Bergmeyer, 1971). The enzymic method however suffers from its sensitivity to any enzymic inhibitor which might be present. Recently Fahey et al. (1981) and Kosower et al. (1983) have introduced a new fluorescent labelling reagent mono-bromo-bimane which labels thiols efficiently and has the added advantage that the derivatives of a wide range of thiols are easily separable by HPLC (Newton et al., 1981). The combination of this labelling and separation procedure then allows identification and accurate quantitation of cellular glutathione levels. MATERIALS AND METHODS Mice of the strain C57 BL/6J were time mated according to the procedures of Whitten (1958) and killed on the appropriate day of gestation. Red blood cells were obtained as previously described (Purdie et aL, 1983). HPLC was performed on a Waters HPLC system employing a reversed phase C18 column. Elution was performed using an isochratic system comprising 95% (10% methanol and 0.25% glacial acetic acid, pH 3.9) and 5% (90% methanol and 2.5% glacial acetic acid, pH 3.9). Glutathione labelling
The glutathione labelling procedure was based on the method previously reported by Kosower et al. (1983) with several major modifications. For each analysis typically 30/zl of red blood cells were employed. Five #1 of the red blood cells were diluted to 3 ml with distilled water and the haemoglobin concentration of the sample determined from the optical absorption measurement at 415 nm employing the extinction coefficient given by Antonini and Brunori (1971). The remaining 25 #1 of red
0.7sl 0..~ u~ LD
Fig. 1. The effect of the volume of red blood cells (Hb/11) used in the assay system, described in the text, on the apparent glutathione to haemoglobin ratio (O) is shown. The value obtained by extrapolation to zero red blood cell volume (73) is also indicated.
blood cells was mixed with 25/zl of monobromobimane in 50% acetonitrile containing 40raM N-ethylmorpholine at pH8.0 to a final concentration of 2ram monobromobimane. The resulting solution was heated to 60'C for 5 rain to allow complete reaction. Excess reagent was then reacted with an excess of/~'-mercaptoethanol (controls using thiol agarose to remove excess monobromobimane as described by Kosower (1983) were shown to produce equivalent results). Then l l/xl of 10% glacial acetic acid and 75 pl of water were added to the sample to precipitate protein. The resulting suspension was centrifuged to remove the precipitated proteins and 5#1 applied to the HPLC column. Glutathione was identified from its retention time after the column had been calibrated with a range of thiol derivatives. This procedure was calibrated in terms of the amount of glutathione present by including glutathione standards in each analysis set.
RESULTS H P L C analysis of the products of the reaction of red blood cells with m o n o b r o m o b i m a n e indicated that glutathione was the only major small molecular weight thiol present in both the adult and embryonic red blood cells from 13 days gestation. In order to ascertain to what extent the labelled product might be co-precipitated with haemoglobin, during the
analysis, variable amounts of haemoglobin were treated by the method described above and the level of glutathione present related to the amount of haemoglobin employed. Figure 1 shows the apparent ratio of glutathione to haemoglobin obtained at various haemoglobin levels. (All dates presented in the following results have been obtained by extrapolation back to "zero" haemoglobin concentration to yield a true value for the glutathione present.) In comparing gluthathione levels between the adult and embryonic stages it is important to note that the procedure required to obtain embryonic samples requires approx 1 hr to complete from the time of killing while the adult sample can be obtained in approx 15 min. Figure 2 clearly shows that the apparent concentration of glutathione in the red blood cells is time-dependent with a considerable decrease occurring in the first hour following killing. Dissection of the embryos in glucose containing saline or maintenance of both embryonic and adult red blood cells in glucose supplimented buffer however clearly prevents the loss of glutathione over this period (Fig. 2). The effect of gestational age of the embryonic sample on the level of glutathione present in the red blood cells was also studied. It was found that no significant difference between the embryonic and adult ratio of glutathione to haemoglobin existed between day 13 of gestation and birth (Fig. 3).
; 0'8 0.6
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Fig. 2. The effect o f aging on the apparent glutathione to haemoglobin ratio is shown for both adult (O) and embryonic (D) samples. Filled symbols refer to data obtained when the cells were maintained in a medium containing 12 mM glucose. The zero time point is also indicated (A), All times are relative to the time of killing.
Fig. 3. A comparison of the glutathione to haemoglobin ratio present in embryonic samples (O) of various gestational age with that seen in adult blood (I-q),
Embryonic glutathione DISCUSSION This microanalysis of the glutathione content of the red blood cells of C57 BL/6J mice yields values for the glutathione to haemoglobin ratios similar to those reported for the red blood cells of adult human (Beutler, 1971). It also highlights some shortcomings of some other reports present in the literature. Most investigators either have not measured values corrected for glutathione decay or else have not reported these corrections. From this present work it is clear that the time delay between killing and analysis is of considerable consequence, yielding values almost 30% too small after 1 hr. It is also apparent that when employing methods which involve precipitation of the protein components of the red blood cell it is necessary to correct results for the co-precipitation of the labelled glutathione. The use of glutathione standards in the absence of added precipitated protein will consequently yield values for the unknown which are too small. To avoid these two problems it is necessary firstly to correct for co-precipitation of labelled glutathione, using data such as that given in Fig. 1, and secondly to correct back to zero time using such data as in Fig. 2 or else collect and incubate the red blood cells in glucose supplemented buffer. In terms of the values obtained for the mouse the adult and embryo (between 13-17 days gestation) they showed essentially identical gluthathione to haemoglobin ratios of 0.6. In both the adult and embryonic mouse glutathione decays quite rapidly but can be maintained by the presence of glucose. It is well known that both the glutathione synthesis, by way of ATP requirement (Meister, 1974) and glutathione reductase, by way of NADPH requirement (Meldrum and Tarr, 1935) require the presence of glucose within the red blood cell. In both cell types it would thus appear that the glycolytic and pentose metabolic pathways are present and active and rely almost exclusively on a source of extracellular glucose. With regard to the developmental pattern in the embryonic red blood cells, the cells have obviously attained their "adult" status by day 13 of gestation in terms of their glutathione to haemoglobin ratio, even though during this period active haemoglobin synthesis is occurring in these cells (Purdie et al., 1983). Although from about day 13 of gestation onwards nucleated red blood cells are replaced by definitive non-nucleated erythrocytes the coincidental values of glutathione to haemoglobin ratios within these two sets of cells means that no change is seen in this ratio during the latter stages of development within the mouse. It would thus appear that the protection of the intracellular haemoglobin and red cell membrane from oxidative damage requires approx 0.6moi of glutathione per tool of haemoglobin irrelevant of the surroundings in which the individual finds itself. Although required for the maintenance of normal haemoglobin function, glutathione concentration can have no role to play in modulating the varied haemoglobin oxygen binding characteristics seen in the embryonic mouse system (Purdie et al., 1983). Acknowledgements--This work was supported in part by a grant from the M.R.C. (New Zealand). We gratefully C.B.P. 83/4B~J
acknowledge the skillful operation of the HPLC by Mrs R. Hill. REFERENCES
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THOMAS BRITTAIN and BRONWYN TOTTLE
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