Determinations of amino acid, cation and reduced glutathione levels in the red cells of Awassi sheep (Ovis Aries)

Camp. Biochem.

Physiol.,

1973,Vol. 46A, pp. 103 to 107. Pergamon

Press. Printed

in Great Britain

DETERMINATIONS OF AMINO ACID, CATION AND REDUCED GLUTATHIONE LEVELS IN THE RED CELLS OF AWASSI SHEEP (OK3 ARIES) ELIZABETH A.R.C.

M. TUCKER,

J. C. ELLORY

and L. KILGOUR

Institute of Animal Physiology, Babraham, Cambridge CB2 4AT, England (Received 22 November 1972)

Abstract-l. Analysis of Na and K in red cells from 126 Awassi sheep revealed 15 individuals with a lowered total concentration of Na + K. 2. These low (Na + K) red cells were found to contain high concentrations of amino acids, and to have a low reduced glutathione (GSH) concentration. 3. Red cells from other GSH-low type Awassi sheep were found to contain normal (Na + K) levels and no significant concentrations of amino acids.

INTRODUCTION IN A SURVEY of six breeds of sheep Tucker & Kilgour (1970) showed that some individuals of the Finnish Landrace and Merino breed had red cells with a deficiency of reduced glutathione (GSH). These GSH low type red cells from the Finnish sheep were found to have significantly lower Na and K concentrations than normal cells (Tucker & Ellory, 1971), and this was associated with the presence in the cells of high concentrations of amino acids, mainly lysine and ornithine (Ellory et al., 1972). In contrast GSH low type red cells from Merino sheep had normal cation levels (Tucker & Kilgour, 1972), and no significant amounts of amino acids. It is well established (Evans, 1954) that sheep red cells fall into two clear genetically determined types with regard to their cation composition, one group having high potassium concentrations of SO-90 m-moles/l. packed cells (HK type) and the other group low potassium concentrations of 20-30 m-moles/l. packed cells (LK type). However, in a more detailed study Evans (1957) suggested that there were four distinct groups of sheep which could be identified from a diagram where red cell K concentrations were plotted against red cell Na concentrations. In particular Evans identified a group of sheep of apparently HK type but with red cell K levels of 60 instead of 80-90 m-moles/l. packed cells, which he called Ke8 type. This type of animal was very rare in British breeds of sheep, but relatively common in Awassi sheep in Israel (Evans, 1966). It has previously been suggested that HK, GSH low type red cells may be the Ke8 type (Tucker & Kilgour, 1970), and the present investigation was carried out to see if the cation and GSH levels in Awassi sheep red cells were consistent with this proposal.

103

104

ELIZABETHM. TUCKER, J. C. ELLORY ANDL. KILCOUR MATERIALS

AND

.

METHODS

Blood samples were taken into heparinized containers from 126 Awassi sheep, and shipped by air from Israel to England. A period of 12-24 hr elapsed between sampling and analysis. Aliquots of whole blood were assayed for GSH by the method of Beutler et al. (1963), using DTNB (5,5-dithiobis-(2-nitrobenzoic acid)). Whole blood haematocrit was also determined. Samples were washed four times by centrifugation in 10 vol. of isotonic MgCls or choline chloride media, buffered with Tris (pH 7.5 at 20°C). Sodium and potassium concentrations were determined by flame photometry on lysates prepared from these washed red cells. Haemoglobin was determined by the cyanmethaemoglobin method (Archer, 1965). Samples of lysates were analysed for ninhydrin-positive zones after electrophoresis on Phoroslides (Millipore Ltd.) as previously described (Ellory et al., 1972). Selected samples were processed for amino acid determinations on a Locarte amino acid analyser.

RESULTS

AND

DISCUSSION

The results for analyses for red cell Na and K levels are presented in Fig. 1. The distribution is similar to previous results for Awassi fat-tailed sheep (Kerr, 1937, analysed by Evans, 1957; Evans, 1966) with individuals falling into two main groups corresponding to the HK and LK type. There are, however, a

110

r

??

I

I

I

I,

I

I

I

I

IO zc 30 40 50 60 70 80 so Na Cm-molesAxed

I,

100 110

cells]

FIG. 1. The distribution of Na and K in the red cells of Awassi sheep.

number of individuals of both the HK and LK type whose red cell Na and K levels are lower than normal, with the sum of cations falling in the range 5590 m-moles/l. packed cells compared with the usual 105-120 m-moles/l. packed cells. In the case of the HK type these individuals should correspond to Evans’ KeS classification.

AMINO

ACID,CATION

AND GLUTATHIONELEVELS

INSHEEPRED

CELLS

105

When samples of red cell hydrolysates were subjected to electrophoresis and stained with ninhydrin, fifteen individuals showed a strong positive reaction, which subsequent amino acid analysis confirmed as being due to the presence of high concentrations of free amino acids in these cells. Detailed amino acid analyses for samples from eight individuals are presented in Table 1. As was previously found in Finnish Landrace sheep (Ellory et al., 1972) ornithine and lysine are the TABLE

~-AMINO

ACID CONCENTRATIONS

4.15 4.42 1.45 1.42 0.59 0.44 0.92 -

Orn Asp Thr Ser Glu GUY

Ala Tyr

Total

AWASSI

SHEEP

Concentration (m-moles/l. packed cells)

Amino acid LYS

IN THE RED CELLS OF GSH LOWTYPE

12.0

1.89 10.5 1.57 1.49 0.76 0.22 0.68 l-04 0.11

3.86 11.4 0.78 1.47 0.96 0.63 -

12.3 6.9 1.22 1.86 1.03 0.95 0.50 1.09 0.51

2.48 13.5 2.02 1.39 0.66 -

10.5 6.13 1.57 1.79 1 .oo -

18.3

19.1

26.4

20.1

22.1

1.04 -

2.78 13.9 1.70 1.48 0.75 0.54 0.60 1.06 22.8

7.49 0.83 1.23 1.79 1.06 0.70 0.80 1.32 15.2

Concentrations recorded as negative were < 0.05 m-moles/l. packed cells.

two predominant amino acids, but the relative proportions of the two amino acids seems much more variable than was previously found. The mean total amino acid concentration for the eight animals was 19.5 f 1.6 (8) m-moles/l. packed cells. The amino acid-containing red cells were of both HK and LK types. However, the sum of Na+ K concentration in these red cells was 83.5 _+1.7 (15) m-moles/l. packed cells, compared with a value of 109.6 _t 1.0 (106) m-moles/l. packed cells for ninhydrin-negatively reacting cells. Since this result could be effected by a difference in cell water, the mean corpuscular haemoglobin concentration (MCHC) was determined in a representative group of ninhydrin positive and negative animals, the values obtained being 34.6 f. 0.3 (9) and 35.8 k O-5 (16) per cent respectively. Thus there was no difference between the two groups. This is in contrast to the observations of Evans et al. (1970) who found Ke6 sheep to have a higher MCHC than normal. The results of determinations of reduced glutathione levels in the Awassi samples are presented as a histogram in Fig. 2. There is a bimodal distribution, with a significant proportion of individuals with low red cell GSH levels. The data are divided according to the presence or absence of high concentrations of amino acids in the cells. All the individuals whose red cells contained amino acids were of the GSH low type, but there were also about an equal number of GSH low type individuals whose red cells did not contain significant amounts of amino

106

ELIZABETHM. TUCKER, J. C. ELLORY ANDL. KILGOUR orn.and

LYS. present

% s

‘o-

Om.andLys.absent

z

GSHconcentration

[m-moles/l.

red cells]

FIG. 2. Histogram of reduced glutathione levels in Awassi sheep red cells. Results are separated into amino acid positive and amino acid negative type red cells.

acids. Table 2 shows the Na+ K content of red cells of both GSH-low types, and it is clear that only the amino acid-containing cells show significantly reduced cation levels. TABLE ~-CATION CONCENTRATIONS IN GSH LOW TYPE RED CELLS Amino acids Present GSH level (m-moles/l. red cells) Na + K level (m-moles/l. red cells)

0.66 + 0.08 (15) 83.5

Results are expressed as the mean f SE.

+ 1.7 (15)

Absent 0.77 + 0.10 (13) 107.6

f 2.3 (13)

(number of animals).

It is concluded that sheep of the Awassi breed show both types of GSHdeficiency which have previously been demonstrated separately in sheep of the Finnish Landrace and Merino breed. One deficiency, previously identified in the Finnish sheep, is characterized by the presence of high red cell ornithine and lysine concentrations and lowered cation concentrations. It seems that this type is identical with Evans’ Ke8 variety, although his classification referred solely to HK type sheep, whilst LK type sheep with similar characteristics have been found in the present study. The other GSH low type, previously found in Merino sheep, is associated with normal cation levels and probably represents a different biochemical lesion. Further analysis of inheritance data and the enzymic pathways involved should lead to a better understanding of the mechanisms involved in glutathione metabolism in sheep red cells.

AMINOACID,CATIONANDGLUTATHIONE LEVELSIN SHEEPREDCELLS

107

Acknowledgements-We would like to thank Uri Katz, Department of Zoology, The Hebrew University, and Professor Ezra Eyal, Agricultural Research Organisation, The Volcani Center, Bet Dagan, for their help in obtaining blood samples from Awassi sheep. REFERENCES ARCHERR. K. (1965) Haematological Techniquesfor Use on Animals, p. 62. Blackwell, Oxford. BEUTLERE., DURON 0. & KELLY B. M. (1963) Improved method for the determination of blood glutathione. J. Lab. clin. Med. 61, 882-888. ELLORY J. C., TUCKER E. M. & DEVERSONE. V. (1972) The identification of ornithine and lysine at high concentrations in the red cells of sheep. Biochim. biophys. Acta 279, 481483. EVANSJ. V. (1954) Electrolyte concentrations in red blood cells of British breeds of sheep. Nature, Lond. 174, 931-932. EVANS J. V. (1957) The stability of the potassium concentration in the erythrocytes of individual sheep compared with the variability between different sheep. J. Physiol., Lond. 136, 41-59. EVANSJ. V. (1966) Red cell electrolytes and haemoglobin. Proc. XIth Congress, Internat. Sot. Haematology, Sydney, 1966, pp. 278-93. Victor C. N. Blight. EVANSJ. V., ROBERTSJ. & AGARN. S. (1970) Delta type Merino sheep: a rare blood type with some erythrocyte characters approaching those found in the camel. Aust. J. exp. Biol. med. Sci. 48, 25-32. KERR S. E. (1937) Studies on the inorganic composition of blood-IV. The relationship of potassium to the acid-soluble phosphorus fraction. J. biol. Chem. 117, 227-235. TUCKERE. M. & ELLORY J. C. (1971) The cation composition of the red cells of sheep with an inherited deficiency of reduced glutathione. Res. vet. Sci. 12, 600-602. TUCKERE. M. & KILGOURL. (1970) An inherited glutathione deficiency and a concomitant reduction in potassium concentration in sheep red cells. Experientia 26, 203-204. TUCKER E. M. & KILGOUR L. (1972) A glutathione deficiency in the red cells of certain Merino sheep. J. agric. Sci., Camb. 79, 515-516. Key Word Index-Awassi

sheep ; red cells; Na and K ; amino acids ; glutathione.