Comp. BIochem Ph)swl~ 1976, Vol 53A, pp 151 co 156 Pergamon Press Prmled m Great Br,ta|n

STUDIES ON AVIAN ERYTHROCYTE METABOLISM--II

RELATIONSHIP BETWEEN THE MAJOR PHOSPHORYLATED METABOLIC INTERMEDIATES

AND OXYGEN AFFINITY OF WHOLE BLOOD IN CHICK EMBRYOS AND CHICKS

R. E. ISAACKS, D. R. HARKNESS, G. A. FROEMAN, P. H. GOLDMAN, J. L. ADLER, S. A. SUSSMAN AND S ROTH

Research Laboratories of the Veterans Administration Hospital and the Department of Medicine, University of Miami School of Medicine, Miami, FL 33125, U.S.A.

(Received 17 February 1975)

Abstract--I The changes in organic phosphates of chicken erythrocytes (RBC) have been determined In relation to the changes in oxygen affinity of whole blood during growth of the embryo and chick

2 2,3-dlphosphoglycerlc acid is the major organic phosphate (42--47%) in the RBC from 15-days of incubation until hatch. The amount of 2,3-DPG during this period IS ~ 4-6 prnoles/cm 3 RBC, decreases abruptly at hatching, and disappears from the RBC within 8 days after hatching.

3. Adenosine trlphosphate (ATP) is the major organic phosphate (45-5070) during the first 5 days post-hatching.

4. The P5o of the whole blood during the last week of incubation and the first 5 days post-hatching correlates best with the amount of ATP in the cells.

5 The effect of inositol pentaphosphate on increasing Pso of the whole blood is more gradual and appears to become of major influence in the chick after 4-5 days post-hatching

INTRODUCTION ERYTHROCYTES of most mammahan species are char- acterized by high concentrations of 2,3-diphosphogly- ceric acid (2,3-DPG). This organic phosphate binds to hemoglobin, substantmlly lowering its affin|ty for oxygen. It is believed that 2,3-DPG functions as an ~mportant intracellular regulator of hemoglobin func- tion (Chanutin & Curnish, 1967; Benesch & Benesch, 1967). On the other hand, nucleated red cells of mature species of birds and some species of turtles are devoid of 2,3-DPG but are charactertzed by high concentrations of an mositol polyphosphate (Rapa- port, 1940; Rapaport & Guest, 1941). Recently the inositol polyphosphate, originally thought to be the hexaphosphate or phytlc acid (IHP), has been more accurately |dentified as the 1, 3, 4, 5, 6 pentaphos- phate (IPP) of inositol (Johnson & Tate, 1969).

In vitro studies have shown that IHP and adeno- sine tnphosphate (ATP) bind to chicken and pigeon hemoglobins and lower the oxygen affinity, presum- ably functioning in a way similar to that of 2,3-DPG in mammalian erythrocytes (Vandecasserie et al., 1971; Ochiai et al., 1972; Vandecasserie et al., 1973). IHP also binds to human hemoglobin and is actually more effective in lowering oxygen affin|ty than is 2,3- DPG (Benesch & Benesch, 1967; Tyuma et al., 1971 ; Janig et al., 1971). The amount of IHP in the red cell of the chicken increases rapidly after hatching (Oshima et al., 1964) and the oxygen affimty of the blood of maturing chicks decreases with age (Hall, 1934). The increase in IHP coincides with, and may be responsible for this observed decrease in the whole

blood oxygen affinity. Recently we have demonstrated that 2,3-DPG, previously considered absent m avian erythrocytes, is not only present but actually is the major organic phosphate in these cells during the week preceding hatching (Isaacks & Harkness, 1975).

The studies reported in this commumcation were designed to provide more complete information on the changes that occur in the organic phosphate com- ponents of the avian red cell and their relationship to changes in whole blood oxygen affinity during growth of the embryo and chick.

MATERIALS AND METHODS

Embryos were obtained from fertde eggs of White Leg- horn hens incubated at 99'5°F in a Humldalre, Model 50, incubator (The Humldaire Co, Wayne St.. New Madison, OH). Eggs were opened at the blunt pole on either the 14th, 15th, 17th, 18th or 20th-day of incubation and the embryo removed. Care was taken to avoid unnecessary rupture of blood vessels Blood samples were withdrawn from the embryos by cardiac puncture with a heparlnized tuberculin syringe. Pooled samples from embryos of the same age were used in each experiment. Blood from 14 and 15-day embryos was drawn into heparin and pooled in 5-6 ml of 8% potassium oxalate which was found to be effective in preventing gelation, a property peculiar to the blood of embryos of this age.

Blood was collected from l, 3, 5, 8, 15, 28 and 36-day chicks (White Leghorn cockerels, Callus domesticus) in heparinized syringes by cardiac puncture and pooled in heparinized tubes. The blood was chdled in an ice bath and transported to the laboratory for immediate use.

151

152 R.E. IshACr~ et al.

The red cells were washed four times with physiologic saline. Capillary packed cell volumes (PCV) were measured on the final suspension and used to calculate the volume of red cells extracted. Phosphorylated metabolic interme- diates were extracted from the washed leukocyte-free eryth- rocytes with trichloroacetic acid (TCA), separated on Dowex I-X8 formate columns using a linear ammonium formate: formic acid (40: 60, pH 3.45) gradlent, and quanti- tated as previously described (Isaacks et at., 1975). Glyceric acid was quantitated by the chromotropic acid assay (Bart- lett, 1959). Oxygen dissociation curves were performed on about 2ml of whole blood at pH 7"4, pCO2 40mm Hg, and 37°C using a modification of the continuous recording method of Longmuir & Chow (1970) as previously do- scribed (Lian et al., 1971).

Hemoglobin electrophoresis was performed on cellulose acetate in a Beckman Model R-101 Microzone Electro- phoresis con using @IM sodmm diethyl barbiturate buffer, pH 8-6. The relative amounts of separated hemoglo- bins were quantitated using a Beckman Microzone Den- sitometer, Model R-110. Cyanmethemoglobin and packed cell volume (PCV) were measured by standard techniques.

RESULTS

Total phosphate and inorganic phosphate of TCA extracts and inorganic phosphate from column fractions

The total phosphate in the erythrocytes from chick embryos was 37 prnoles/cm a RBC at 14 days of incuba- tion and decreased to 23 #moles/cc RBC immediately before hatching (Table l). A/fter hatching the total phosphate level of the chick erythrocyte was 27- 33 #moles/era a RBC with possibly a slight increase around 8 days post-hatch (Table l). The Pi content of the TCA-cxtracts of erythrocytes from 14 day embryos was approximately 24 pmoles/cm a RBC, (64% of total phosphate), and this gradually decreased to a level of approximately 6 #moles (~ 20% of total phosphate) ~in erythrocytes from 36-day chicks (Table l). Quantitation of Pi after column fractionation

revealed much lower levels of Pi: approximately 6.5gmoles/cm s RBC in the 14-day embryo and 1.4 #moles in erythrocytes from 36-day chicks. This represents only about 18 and 4% of the total phos- phate, respectively (Table 1).

AMP, ADP, ATP and other nucleotides

AMP remains fairly constant, between 0.2 and 0.5 gmoles/em 3 RBC, during incubation and early de- velopment of the chick, accounting for less than 1.0°/0 of the total cell phosphate (Table 1). ADP is present in the red cell from the 14-day embryo at a level of 1-9 pmoles/cm "~ RBC and generally decreases to 0"35#molcs/cm 3 RBC in the red cells from 36-day chicks (Table 1). These levels represent 10-5 and 2"3% of the total cell phosphates, respectively.

The quantity of ATP in the erythrocytes of 14-day embryos is 3"8gmoles/crn 3 RBC, decreases to 1-8 pmoles/em a RBC at 18 days of incubation, begins to increase at 20 days of incubation, reaches a level of 4.8/zmolcs between 3 and 5 days post-hatching and then decreases to 2.0 #moles/era a RBC m erythrocytes from 36-day chicks (Table 1). ATP comprises 31% of the cell phosphate at 14 days of incubation, 45- 50% of the cell phosphate the first 5 days post-hatch- ing, and decreases to 16--20% of the cell phosphate at 36 days (Table 1).

The ratio of AMP:ATP during incubatmn ]s between 0"1 and 0"2 whereas after hatch this ratio is approximately one-half this value (Table 1). The ratio of ADP:ATP remains fairly constant (~0"5) during incubation and falls after hatch to a ratio of 0.15 to 0.2. The ratio of ADP:ATP after hatch agrees well with the ratio observed in mammalian erythro- cytes. The reason for the higher ratios during incubation is not readily apparent.

Two unidentified nucleotides, "XI" and "X2", appear in erythrocytes of the 14-day embryo (Fig. 1).

ex l~ac t

TABIJ~ 1

DJJt~'J.but.:Lon o f the NaJor lq lo~hox 'yla t ;ed He'cabollc

~[ntezmsedlatam I n Bzythzocytes o f C~t:Lck Z~b~',/os

mad (~Ickm (uaoles ~ ; ; N~:)

co lv :m f :ac~Lon

14 - r ~ 23.95 37.14 0.42 6.$7 1.94 +--3.89 +4.68 +.03 +0.55 +O.O3

15 - r ~ 14.8 32.65 O. 43 S. 87 1.30

17 - rm 10.52 25.15 0.47 4.19 0.83

18 - r ~ 7.82 24.39 0.17 3.84 0.58 +._0.73 +0.78 +0.01 +0.08 +0.08

20 - r ~ 8.00 21.50 0.33 3.00 0.78 _+0.47 +1.50 +0.14 +..0.$6 +._0.21

1 - r " 12.97 27.74 0.31 2.61 0.72

3-DC 9.31 32.27 0.32 2.31 0.64

S-DC 14.52 33.75 0.26 2.41 0.94

8-DC 7.53 40.31 0.38 2.31 0.68

1S-DC 7.20 31.22 0.27 1.37 0.29

28-DG 7.70 29.04 0.22 1.18 0.24

36-DC 6.17 31.40 0.25 1.36 0.35

2t3-Dt~G ZPP o thez 2 l~cov. • ]~cov.

3.83 1.57 0.32 8.96 35.0 64.S +o.64 _+o.o6 _+0.20 +_1.o5 +-3.8

2.85 4.02 0.38 - 31.2 95.5

1 • 58 5.88 0.42 0.36 25.3 100.5

1.80 5.10 0.48 1.04 23.3 95,8 +_o.48 +._0.21 _+0.0~ ~ . s o _+o.5

1 • 65 4 * 32 0.79 1 • 12 23 * 2 107 • 1 _+0.33 ~.57 +_o.ss ~.9o +~.3

4.34 1.13 2.15 2.02 27.3 98.5

4.84 0.58 1.42 2.48 29.2 90.4

4.84 2.69 2.88 35,5 105.1

4.01 3.47 2.93 36,4 90.2

2.66 3.25 1.30 26.5 84.8

1.SS 3.41 0.94 24.5 84,5

~.00 3.66 1.11 27. 7 88.2

Of ch ick kn " l "ys. Pl�"uz-es re£ex: to m a n

"X2m o

l r ~ 1manta age o f eabz,jro i n (l~Wn ~ 0c R m ~ + S O .

2 Z n o l ' ~ " ~ l ~ w ]mAD, ~ m ~ ~wo ueMden~L££ed n~cleotJ.mem mXlm

Studies on avian erythrocyte metabolism 1$3

pmol~/ml

1, I ADP

_E :2 n I*TP/

l 0

i °' 0 6

0 4

0 2

0 I f I I I i I I I I 0 20 40 60 80 100 120 140 160 180 200

FRACTIONS (5 ml)

Fig. 1. Column chromatogram of TCA extract of 5'58 cm a RBC from 130 14-day embryos The chromatogram was developed using a linear gradient composed of 500 ml dis- tilled water and 500 ml ammonium formate (60 parts of 5N formic acid to 40 parts 5N ammomum formate, pH 3.45). Narrow solid line shows absorbance at 260 nm, wide sohd line shows/zmoles Fh/ml of fraction and the broken

hne indicates #moles glycerate/ml fraction

Nucleotide "X~" is still present in erythrocytes from 18-day embryos while nucleotide "X2" has disap- peared (Fig. 2). The second unidentified peak elutes in a position similar to the iron-ATP complex for- merly designated "AXP" (Bartlett, 1970). We have not yet attempted to identify either "Xl" or "X2".

Changes in 2,3-dzphosphoglycerate 2,3-diphosphoglyceric acid was detected initially in

the TCA extract of erythrocytes from 18-day chick embryos by observation of a phosphate peak corre- sponding in elution position under these column con- ditions to standards of this compound (Isaacks et al., 1975). The identity of this material has been further verified by obtaining the appropriate color reaction with the chromotrop]c acid assay for analysis of gly- cerate (Bartlett, 1959). The phosphate to glycerate ratios are 2:1 and the neutralized acid-soluble extracts stimulate PGA mutase activity in an enzyma- tic assay for 2,3-DPG (Towne et al., 1957; Isaacks & Harkness, 1975).

The level of 2,3-DPG in erythrocytes from 14-day embryos was 1.6wnoles/cm 3 RBC. It reaches 5-- 6/anoles/cm 3 RBC between 17 and 18 days of incuba- tion and maintains that level until just before hatch- rag. Immediately after hatching a sudden drop in 2,3- DPG occurs (Table 1; Fig. 1; Fig. 2; Fig. 3; Fig. 5). Within 3 days post-hatching, 2,3-DPG has de- creased to values below those detectable by the tech- niques employed in these studies. The amount of 2,3- DPG in the erythrocytes from 17 and 18-day embryos represents 42-47% of the total cell phosphate. It is the major organic phosphate in the erythrocytes of chick embryos from day 15 of incubation until hatch.

Changes in inositol pentaphosphate Very low concentrations of IPP are present in the

erythrocytes of embryos through 18 days of incuba-

.umoles/ml

19

16

14

I O4

0 2

0

I I ATP

P,

I I I

~ 3 ~

I I I l l I I 0 20 40 60 80 100 120 140 160 180 2(~

I~CT IO I~ (5 ml)

Fig. 2. Column chromatogram of TCA extract of 7.13 cm 3 RBC from 40 18-day embryos. The chromatogram was de- veloped with the same buffer system and gradient de. scribed in Fig. 1. Narrow sohd hne, wide sohd line, and

broken line notations are the same as in Fig. 1.

tion (Table 1; Fig. 1; Fig. 2). Just before hatch IPP begins to increase and continues to increase during the first few days post-hatching (Fig. 4) reaching a level of 3.5 ~noles/cm 3 RBC on day 8 (Table 1 ; Fig. 5). IPP represents only approximately 4% of the phos- phate of erythrocytes of 14-day embryos and almost 60°//0 of the phosphate of erythrocytes of 36-day chicks (Table 1).

Changes in hematocrit, hemoglobin, and Pso The packed cell columes, hemoglobin con-

centrations, and whole blood Pso'S at various ages of the embryo and the chick are given in Table 2. The values for Pso reflect an increase in oxygen affinity from 23"9 mm Hg in whole blood of 13-day embryos to 28.2mm Hg in whole blood of 15-day embryos (Table 2; Fig. 5). The Pso then decreases to 21"5 mm Hg in 18-day embryos and increases to 23.1 mm Hg in 20-day embryos (Table 2; Fig. 5).

umoles/ml

18

16

14

[ 12

i° , 0 4

0 2

0 I

0

ATP

Al~ o

3 DiG

OTP

I I I I I I I I 20 40 60 80 100 120 140 160 180 200

F R A ~ (5 ml)

Fig. 3. Column chromatogram of TCA extract of 5.17 cm 3 RBC from 21 20-day embryos. The chromatogram was de- veloped with the same buffer system and gradient de- scribed in Fig. I. Narrow solid line, wide solid line, and

broken line notations are the same as in Fig. I.

154 R.E. ISAAC,S et al

jamole~/ml

18

16

14 ATP

12

>. 10 IPP

~ 0 6 ADP

0 I I I I I I 100 120 140 160 180 200

FRACTIONS (5 roll

Fig. 4. Column chromatogram of TCA extract of 4'71 cm 3 RBC from 12 5-day chicks. The chromatogram was devel- oped with the same buffer system and gradient described in F~g. I. Narrow sohd line and wide solid line notations

are the same as in F~g. 1

Thereafter the whole blood Pso increases abruptly to 37.1 mm Hg in 5-day-old chicks, plateaus for several days and then further increases to 42.8 mm Hg m 36-day chicks (Table 2).

The changes in relative amounts of the embryomc (Hb-E), adult major (Hb-A), adult minor (Hb-D), and the trace hemoglobins are shown in Table 3. Hb-E comprises 8.3% of the hemoglobin in the erythrocytes of 13-day embryos. It remains around this level at all stages of embryomc development and through the first 6 or 7 days posthatching and then decreases to 1-5% in the 36-day chick (Table 3). The ratio of hemoglobin A to D remains approximately 2:1 throughout development of the embryo and m the young chick. Similar distributions of these hemoglo- bins have been described by Washburn (1968).

DISCUSSION

Avian blood has a relatively low oxygen affinity when compared with blood of most mammals. The Pso of chicken blood which is the h~ghest of those

~umoles/cc RBC P50

,0 / ° \T 4 0 w ~ / / " ~ . IPP 35

~o ;~ Yl o / \o ~o

# I o zo : "\- o / / , / \o

I 0 20

0 I I s 2 3

14 16 18 20 0 2 ,4 6 8 15 22 29 36

HATCH DAYS

Fig. 5. Correlation of whole blood Pso with 13-DPG, IPP, and ATP content of erythroeytes from chick embryos and

young chicks.

~ 2

CHANGZS IN PCV, HEMOG3~BIN CONTENT OF WHOLE BLOCO, ~qD

P50 OF WHOLE BLOOD DURING INCUBATION AND THE EARLY

D g V l ~ O p I ~ OF THE C3tlC~ 1

Age PCV Hemoglobin P50 (per cen t ) ( I g / m l )

13-DE 23.9+--7.9

1 4 - ~ 17.9+--3.1 63.5+_2.6 27.7+_2.6

15-DE 19.3+--3.6 80 .0 28.2+_2.8

16-DI 20 .1+0.6 81.5 26.5+-0.4

17-CIE 22 4

18-DE 22.8+--4.1 59.2+_14 3 21.5+._O.5

20- IX 27 1+--1.0 71.3+_26.2 23.1+-0.9

I-DC 24.2+_2. I 85.1+--4.7 33.7+--2.5

2-'pc 23.3+-1.0 81.1+_e. 3 32.6+-1.0

3-DC 23.2+_1.4 82.7+_4.1 36 • 0+-1 • 3

4-DC 24.2+__1.0 78.8+--5.4 37 .2+0.9

5-De 25.0÷-1. S 84.1+--6.3 37.1+O. 2

6-OC 25.1+2.2 81 • 6+5.8 -

7-DC 25.3+_1 3 61.4+_4.8 36.1

8-DC 28.0+_3 0 94.4+--12.5 36.5+-0.7

15-DC 29.5+-3.5 108.4+-0.8 41 • 3+3.9

22-DC 28 • 0+-0.7 81 • 9+-11.5 43.9+--2.5

29-DC 30.5+-1.4 92 • 5+4.5 45 • 0+_6.2

36-DC 27.3+_3.2 85 • 5+_9.6 42 • 8+-1 • 4

adult 40.5F3.3 1 3 4 . 9 ~ 1 o . I a6.8,+9.6

IDE means age of embryo in days and IX: means aqe of

chicks in days. Figures refer to mean values + S.D.

TABLE 3

PER ~ CONCENTPJ%TION OF THE VARIOUS HEMOGLOBIN BR~S 1

~Je t ~ - E I ~ -A : ~ - 0 Trace

1 3 - ~ (10) 8 .3 60.7 28.1 2 .9

14 - I~ (13) 5 .8 61.4 31.O 1 .8

I5-~lB (3) 5 .1 64.2 30.0 0 .5

16-08 (2) 6 5 63.S 26.9 1 .1

18 -rm (12) 7 .4 66.1 24.8 1 .9

20-ON (4) 5 .5 66.9 26.3 0 .9

1-0C (10) 4 .6 57.6 34.9 2 .4

2-DC (5) 7 .5 62.6 27.9 2 .0

3-1X: (7) 6 .8 66 .5 25.2 1.7

4-DC (5) 7 .0 68.0 21.5 3 .5

5-DC (7) 6 .0 60.5 32.3 1 .3

6-0C (6) 7 .4 59.9 29.6 3.2

7-DC (6) 8 .5 71.2 22.6 0 .7

8-DC (10) 3.7 62 l 32 .8 1 .5

15-DC (4) 1.3 57.7 40.6 1 .4

22-DC (4) 1.O 58.5 38.9 1 .8

29-DC (4) 1 .2 61 .6 36.4 1.1

36-1X~ (4) 1 .5 59.6 37.4 1 .6

~ l u l t (4) 2 .4 87 .0 30.1 0 .9

1Dig ~ a n s a g e o f embryo i n d a y s a n d IX: means a g e o f c h i c k i n d a y s .

~ - E i n d i c a t e s e - ~ r y o n i c he~ocj lc~bin, ~ - A i n d i c a t e s a d u l t o r m a j o r

h e m o g l o b i n p a n d ~ - D £ n d ~ c a t : s l a d u l t g d ~ o r h e m o g l o b i n . The n t ~ b e r

Of o b s e r v a t i o n s £nvolved l a shmm I n paren theses a f t e r the age no-

ta~Lon.

Studies on avian erythrocyte metabolism 155

reported in birds, gradually increases with age (Mor- gan & Chichester, 1935; Hall, 1934). In both mam- mals and birds, so far as known, the blood of the embryo has a higher oxygen affinity than that of the adult. In mammals this difference in oxygen affimty between maternal and fetal blood is thought to facili- tate transport of oxygen across the placenta. Presum- ably the higher affinity of chick embryo blood facili- tates transport of oxygen across the allantoic mem- brane The decrease in oxygen affinity of the blood as chick matures has been attributed m one report to the replacement of the molecular species of embryonic hemoglobin with adult hemoglobin (Hall, 1934). However, our data (Table 3), as well as data from other studies, does not indicate a change in hemoglobin distribution during this period of suffi- cient magnitude to account for the observed abrupt decrease in oxygen affinity. Huisman & Schillhorn Van Veen (1964) reported that Pi in the red cells of embryos decreased with age and continued to de- crease in the chick. They suggested that the Pi level in the red cell might regulate the oxygen affinity of hemoglobin in the developing embryo and chick. Our data (Table 1) verify the findings of Irving & Cos- grove (1970) that attempts to measure P1 in the pres- ence of organic phosphates, particularly IHP and pre- sumably 2,3-DPG, as well as other labile phosphate esters, gwes erroneously high values for PL This per- haps explains why the earlier workers considered the Pi content to be of such importance.

Our own data reported here indicate that the most plausible explanation for this increase in Pso with age is the accumulation of IPP The data are in agreement with the known effect of IHP upon oxygen affimty of hemoglobins (Vandecasserie et al., 1971 ; Ochial et al., 1972; Vandeeasserle et al., 1973) and the previous reports of increasing IHP with age after hatching (Oshtma et al., 1964).

It would appear from our data, however, that the very rapid increase m Pso which occurs precisely at hatching correlates best with the very abrupt rise in cellular ATP and that the effect of IPP is more gra- dual, becoming of major influence after the chick is 4 or 5 days of age. Our data on changes in Pso and ATP level during the last week of incubation agree with previous reports (Bartels et al., 1966; Misson & Freeman, 1972).

The data on Pso values m the younger embryo ms of considerable interest because the Pso is lowest at a time when 2,3-DPG is very high. The Pso is highest at 15 days incubation at a time when ATP is high. In fact, throughout the period of embryonic develop- ment just as in the young chick, the whole blood Pso correlates best with the amount of ATP in the cells and is seemingly httle affected by the extraordi- narily high amounts of 2,3-DPG. This observation seems in contradiction to the effect of 2,3-DPG on oxygen affinity in mammahan red cells. If it does not alter the oxygen affinity in these cells, it may be that the 2,3-DPG is compartmentalized and unavailable for binding with the hemoglobin molecules. Studies on the distribution of organic phosphates within these cells are now underway in our laboratory.

The dramatic switch from 2,3-DPG as the predomi- nant organic phosphate of erythrocytes of the chick embryo to IPP shortly after hatching suggests an

abrupt activation and inactivation of genes. During the period immediately before and after hatching the embryo passes from the fetal state through the parafe- tal state into the postfetal state. The parafetal period begins when the beak of the embryo breaks the mem- brane to the air space and terminates when the chick perforates the blunt end of the egg shell with its beak. The parafetal period lasts 24 hr or more and is the period during which allantoic circulation dechnes and pulmonary respiration commences. The compositaon of the air space in which the chick respires initially mn the parafetal period is exceedingly high in carbon dioxide content, 9-11~, and the oxygen content is less than half atmospheric, 8-9~o (Romijn & Roos, 1938). Carbon dioxide is a more efficient respiratory stimulus than hypoxia and is thought to initiate breathing (Windle et al., 1938; Freeman & Misson, 1970). Inhalation of the high content of carbon diox- ide in the air space causes increased respiratory muscle contractmns. It is beheved that this results in the embryo perforating the egg shell (Windle & Barcroft, 1938). Therefore during hatching, the embryo passes suddenly from an environment of low pO2 and high pCO 2 to one of normal oxygen and very low CO2 tensions. During this same period, oxygen consump- tion increases by 125-150%. These changes coincide with a dramatic rise in the arterial pO2 from about 20 mm Hg in the mature embryo to almost 110 mm Hg in the neonate (Freeman, 1962; Freeman & Mis- son, 1970).

We have proposed prewously in a brief communi- cation that the sudden changes in oxygen tension that result from the change-over from passive diffusion into the egg to active respiration in the newly hatched chick may trigger these events (Isaacks & Harkness, 1975). It is also possible that these observed changes m distribution of cellular phosphates which occur at this time could be related to the abrupt changes in pCO2.

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BARTLETT G R (1970) Patterns of phosphate compounds m red blood cells of man and animals In Advances m Experimental Medicine and Biology (Edited by BREWER, G. J.), Vol. 6, pp. 245-256 Plenum Press, New York.

BARTEt~ H., HILLER G. & REtNHARDT W. (1966) Oxygen affinity of chicken blood before and after hatching Resp. Physml. 1, 345-356.

BENESCH R & BENESCH R. E. (1967) The effect of organic phosphates from the human erythrocyte on the allosterlc properties of hemoglobin. Biochem blophys. Res Com- mun 26. 162-167

CHANUTIN A. & CURriSH R. R. (1967) Effect of organic phosphates on the oxygen equlhbnum of human eryth- rocytes. Archs. Biochem. Biophys. 121, 96-102.

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FREEMAN B. M. & MISSON B. H. (1970) pH, p02 and pC02 of blood from the foetus and neonate of Gallus domes- t~cus. Comp Biochem. Physiol. 33, 763-772.

HALL F. G. (1934) Hemoglobin function m the developing chick. J. Physiol., Lond. 83, 222-228.

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