British Journal ofHaematology, 1971, 20,263.

Globin Synthesis in Sideroblastic Anaemia

I. a AND B PEPTIDE CHAIN SYNTHESIS

J. M. WHITE, M. C. BRAIN AND M. A. M. ALI

Medical Research Council Group on Haernolytic Mechanisms, Royal Postgraduate Medical School, London, W. 12

(Received 3 I July 1970; acceptedfor publication 21 August 1970)

SUMMARY. The in vitro synthesis of the a and B peptide chains of globin have been measured in I I patients suffering from sideroblastic anaemia (four congenital and seven idiopathic acquired). In the 10 patients who were anaemic the findings were similar. The synthesis of the globin chains was found to be defective in two respects; firstly, synthesis was asynchronous with an apparent deficiency of the synthesis of a chains, and, secondly, a large proportion of both u and /3 chains synthesized were not associated with haem but were free in the cell in the form of c$ dimers. Addition of haem to the incubation mixture greatly stimulated the synthesis of both chains, re- moved the free dimer pool and appeared to stimulate a chain synthesis. The finding that a large proportion of the c1 and chains synthesized are not associated with haem but are free within the cell as a dimer pool, is very strong evidence that the underlying defect in sideroblastic anaemia is haem deficiency. The defective syn- thesis of a chains relative to B chains is as yet unexplained but may account for the low haemoglobin A, associated with this condition.

The sideroblastic anaemias are a heterogeneous group of disorders characterized by dyshaemo- poiesis, a population of hypochromic red cells in the peripheral blood and ring sideroblasts in the bone marrow. The anaemia may be inherited or acquired, the latter being either primary, or secondary to an underlying disease ( M o b & Hofirand, 1968). Defective haemoglobin synthesis, as judged by the red cell hypochromia, despite excess iron in the erythroblasts, would seem to be the characteristic feature of sideroblastic anaemia. The finding of diminished activity of one or more of the enzymes responsible for haem synthesis (Heilmeyer, 1967; Vogler & Mingioli, 1967; Rothstein et al, 1968, 1969) had led to the con- cept that the defect lies in the synthesis of haem rather than globin. Other reports, however, have failed to confirm this (Vavra & Po% 1967).

Globin synthesis in sideroblastic anaemia has not been studied in detail, yet there are two reports which indicate that it may not be normal: first, Heller et a1 (1965) reported that the incorporation of [14C]glycine into globin in vivo was subnormal in a patient with sideroblastic anaemia as compared with a normal control, and secondly, Reed & Mollin (1968) reported that the percentage of Hb A, was low in several patients with sideroblastic anaemia but that it returned to normal if they responded to therapy.

The purpose of the present study was to obtain evidence for or against defective globin synthesis in sideroblastic anaemia. The in vitro incorporation of ['Hlleucine into the u and

263

264 J M. White, M. C. Brain and M. A. M. Ali

chains of globin by reticulocytes of several patients with sideroblastic anaemia is reported. Preliminary results have already been published (White et a!, 1969).

Case

___-

10 I1

PATIENTS AND METHODS

Patients. The relevant clinical and haematological features of the 11 patients studied arc shown in Table I. Four patients were suffering from hereditary sideroblastic anaemia and seven from the primary acquired variety. The patients’ illness fulfilled all the diagnostic criteria of sideroblastic anaemia; a hypochromic population of red cells in the peripheral blood, high serum iron, saturated serum iron binding capacity and numerous ring sidero- blasts in the bone marrow associated with greatly increased bone narrow iron stores. Nonc of the patients was found to have an overt underlying disease, and all were unresponsive to pyridoxin and folic acid therapy with the exception of Case 11 who had responded to pyri- doxin and was in remission at the time of investigation.

TABLE I. Haematological values of patients with sideroblastic anaemia

Sex

I M 2 M

3 M 4 M 5 F

7 M 6 F

8 F 9 F

F M

Classification

Hereditary Hereditary Hereditary Hereditary Idiopathic acquired Idiopathic acquired Idiopathic acquired

Idiopathic acquired Idiopathic acquired Idiopathic acquired Idiopathic acquired

Hb(g/~oo ml)

8.9

8.6

7.9 7.0 8.8

9.2 8.0 8.5

13.2

11.0

11.0

M C H C (%I

30 31 28 30 29 29 30

28

30 29 3 1

Retics (%I

6.4 3 .O

2.5 3 .o

3 .o

-

2.0

2.1

2.0 2.0

4.6 1.5

-

Hb Az I Specialfeatrrres (%I

O.’ I } Siblings 0.9 1.0

0.9 0.8 I .o 1.1

0.6 0.9 0.9 2.0

Elliptocytosis

Elliptocytosis, splenectomy

Pyridoxitie-respon- sive. in remission

Haematological values are those at time of study.

The six patients used as controls were all suffering from an autoimmune haemolytic anaemia (AIHA), in two instances secondary to an associated lymphoma. They all had normoblastic erythropoiesis, with no ring sideroblasts.

Routine haevnatological investigations were carried out as describcd by Dacie & Lewis (1968). The percentage of Hb A, was determined by cellulose acetate electrophoresis (Marengo- Rowe, 1965), using tris EDTA buffer, pH 8.7.

The level OfHb A, in the hypochromic and normochromic red cell populations were measu- red in three patients. The hypochromic and normochromic cells were separated into two distinct bands by differences in density, using centrifugation in pthalate oil SG 1.070 (Danon et al, 1966). The two populations of cells were freed from oil by continuous washing on filter paper (Whatman ‘separa’ grade D.H.C.) with isotonic phosphate buffered saline, pH 7.4, then lysed and the percentages of Hb A, determined.

Globin Synthesis in Sideroblastic Anaemia 265 a and p chain synthesis by normal and sideroblastic anaemia reticdocytes isolated jiom whole-cell

yreciyitatedglobin. Forty millitres of venous blood from the sideroblastic anaemia patients and controls were collected into heparin (0.5 mglml). After washing three times in isotonic saline, 10 ml of packed cells were centrifuged at 12 ooo g for 45 min and the top 0.5 ml removed for incubation. The reticulocyte count in the sample taken for incubation was usually three times or more that of the uncentrifuged blood. The incubation mixture used was similar to that described by Lingrel & Borsook (1963) except that leucine was omitted from the amino acid mixture and only 0.5 ml of cells was added. The proportions of plasma and other components were adjusted to : 3 .o ml of plasma reagent mixture, 0.1 ml of ferrous ammonium sulphate and 0.1 ml of glutamine. The mixture was preincubated at 37°C for 30 min in 10 ml graduated centrifuge tubes following which it was centrifuged at 12 ooo g for 10 min and 2 nil of the supernatant removed. IoopCi of [3H]leucine in I ml of the plasma reagent mixture were added to the cells and the incubation continued at 37°C for 60 min. At the end of the incubation time the cells were washed three times in isotonic saline and added immediately to 20 vol of 2% acid acetone at - 20°C to precipitate the globin. The globin was washed three times in acetone, dried in ether and stored at - 20°C.

In order to ascertain that when whole cells were added to 20 vol of acid acetone all the globin chains in the cells were precipitated and none remained in solution, the following experiment was carried out. After a 60 min incubation with [3H]leucine as described, the cells were washed with 10 vol of isotonic saline until no significant radioactivity was detected in the supernatant (usually after five washes). The whole cell sample was then added to 20

vol of acid acetone, and the precipitated globin removed by centrifugation. The supernatant was then added to an equal volume of acid acetone at - 20°C and left to stand at this tempera- ture for 60 min. At the end of this time no further precipitation could be detected. The specific activity of 1.0 ml of this mixture was determined and the remainder was dialysed in 8/32 ‘viskin’ tubing for 24 hr at 4OC against water and the specific activity again determined. Ninety-eight per cent of the radioactivity originally present in the supernatant was removed by the dialysis indicating that it was not associated with whole (non-dialysable) peptide chains. From this experiment it is reasonable to conclude that the addition of whole cells to a 20 vol of acid-acetone results in virtually complete precipitation of all the globin chains present.

The a and B peptide chains of the globin were separated on columns of Whatman CM 23 using 8 M-urea/mercaptoethanol buffers, according to the method of Clegg et a! (1966). The whole of each chain was collected and dialysed against four changes of 5 litres of 0.5% formic acid for 48 hr. The volume and OD at 280 nm of each chain was measured and a I ml sample was added to 10 ml of liquid scintillation fluid (Bray, 1960) and counted in a Beckman Ls-250 liquid scintillation counter. The total counts incorporated into each chain was calculated from the total protein content and the specific activity of that chain. The total counts in the a chain were then expressed as a ratio to the total counts in the p chain (a :/3 ratio).

a and f l chain synthesis by normal and sideroblastic reticulocytes isolatedfrom purified haemoglobin. Cells were incubated with [3H]leucine for 60 min as described above and then divided into two equal samples. One sample was added directly to acid acetone, the globin precipitated and the total counts incorporated into the separated a and /l chains were determined. The other sample was washed three times in isotonic saline, lysed at 0°C by the addition of 4 vol of

266 J. M. White, M. C. Brain and M. A. M. Ali

a 0.005 M-MgCl, solution followed 30 s later by I vol of 1.5 M-sucrose containing 0.15 M-KC~ as described by Lingrel & Borsook (1963). The stroma was then removed by centrifugation at 20 ooo g for 15 min. The haemoglobin from the lysate was purified according to the method of Winterhalter et al (1969). After dialysis of the lysate for 10 hr against 0.01 M phosphate buffer, pH 7.55, it was applied to a 20 x 2 cm column of Sephadex CM 50, equilibrated with the same buffer. The haemoglobin was eluted off the column with 0.01 M phosphate buffer, pH 7.6, until no further protein was detected at OD 280 nm. This chromatographic technique allows only soluble haemoglobin to elute off the column, any free globin chains not associated with haemoglobin being retained. The whole of the eluate was concentrated by ultrafiltration for 10 hr prior to precipitation of the globin and separation of the ct and f l chains. The total counts in the chains isolated from the purified haemoglobin were then compared with the total counts in the chains prepared from globin from the whole cells.

Efect of haem on u and /? chain synthesis in sideroblastic anaemia. To study the effect of the addition of haem on the synthesis and assembly of globin chains the following experiments were set up.

0.652 mg of Haemin* were dissolved in 0.05 ml of I N - N ~ O H to which was added 0.85 ml of substrate plasma and 0.05 ml of Tris buffer. The pH of the mixture was then corrected to 7.4 by the addition of I N-HCl. 0.5 ml of the haem solution (conc. 10- mol/l) were then added to 4 ml of incubation mixture plus cells and the whole preincubated for 30 min at 37°C. Control tubes containing 0.5 ml of substrate plasma were set up and also preincubated. After 3 0 min IOO pCi of ['Hlleucine were added and the incubation continued for 60 min. After this time the mixtures were divided into two equal parts, one half added as whole cells to acid-acetone and the other half lysed and the haemoglobin purified. The effect of the presence of haem on the total counts in the tl and f i chains isolated from whole-cell precipitates and from purified haemoglobin were then determined and compared with the total counts of the ct and fl chains isolated from the control tubes.

Sephadex GIOO chromatography of lysates from sideroblastic anaemia retintlocytes. In another series of experiments a sample of the lysate from an incubation mixture was added to 160 x 3 cm columns of Sephadex G-IOO equilibrated with 0.2 M tris HC1 buffer, pH 7.8. The columns had been calibrated with blue dextran, serum albumin, cytochrome oxidase and ['Hlleucine. The haemoglobin and non-haemoglobin radioactive fractions were eluted off the column with the same buffer over 24 hr, collected in 6 ml fractions and the haemoglobin (OD 540 nm) and protein concentration (OD 280 nm) of each fraction measured. The radioactivity of each fraction was determined by liquid scintillation counting with the use of an internal standard of [ 'HIHexadecane to correct for quenching.

RESULTS

Percentage and Distribution of Hb A, in sideroblastic Red Cells The Hb A, level ofwhole blood in the 10 patients with sideroblastic anaemia on 36 different

estimations ranged from 0.5 to 1.3 ( 5 o.I)%. This is significantly different (P< 0.001) from the level of 1.5-2.5 ( ~ o . I ) % found in 50 normal patients.

The level of Hb A, in the least dense (hypochromic) population of cells in three patients * Haemin (Haematin hydrochloride), British Drug House, Poole, England.

Globin Synthesis in Sideroblastic Ahaemia 267

was 0.24.8 (+ o.I)% whilst that of the most dense (normochromic) population of the same patient was 1.5-1.9 (-+o.I)%. These observations confirm the findings of Reed & Mollin (1968) and indicate that the abnormality in haemoglobin synthesis which results in a low Hb A, resides in the hypochromic population of the red cell in sideroblastic anaemia.

Disease

AIHA (lymphoma) AIHA AIHA (lymphoma) AIHA AIHA A H A

Total a and j Chain Synthesis in Control (Non-sideroblastic) Reticdocytes The ratio of total counts incorporated into the a chain to total counts incorporated into the chain (a :/3 ratio) in the reticulocytes of six control (non-sideroblastic) subjects after 60 min

incubation with [3H]leucine was approximately unity (range 1.00-1 -05, mean 1.02; Table 11).

a

2663 16 347008 565225 162150 284600 111174

TABLE 11. Incorp~rationof[~H]leucine into a andbchains of normal reticulocytes (whole-cell precipitation)

Decrease in counts (%)

a B

5 3 2 6 5 4

-

Case

Ratio a:B

1.01 I .07 I .04

I

Whole-cell ppt; total counts

(CP4

a B

347008 334720 565225 548103 284600 269680

Total counts I (CPm)

Purified Hb; total counts

Ratio (CP4 a:p

a B

1.04 329657 324678 1.03 553920 515216 1-05 271220 260371

B

248540 334720 548103 IS7500 269680 110180

-

Ratio a:B

1.03 1.04 1.03

1.05 I .02

I .oo

TABLE 111. Incorporation of [3H]leucine into u and B chains of normal reticulocytes isolated from whole-cell precipitates and purified haemoglobin

These ratios are in agreement with those reported previously from this laboratory (White & Brain, 1970) and other workers (Bank & Marks, 1966, 1969; Bargellesi et al, 1968; Modell et a!, 1969) and indicate that in the normal reticulocyte both globin chains are synthesized in equal amounts.

In three of the control patients the total count in a and P chains separated from globin prepared by whole cell precipitation was compared with the total counts in these chains obtained after the haemoglobin had been purified on Sephadex CM-SO (Table 111). Separa- tion and purification of haemoglobin did not result in a significant fall in counts in either chain, and the ratio of counts in the a chain to those in the f i chain was unaltered.

C HABM.

268 J. M. White , M. C. Brain and M. A. M. Ali

Sephadex CM-50 is known to retain free chains (Winterhalter e t al, 1969) which are not associated with haemoglobin. Therefore the finding that in normal reticulocytes the radio- active chains are nearly all associated with haemoglobin indicates that as the chains are synthe- sized they rapidly combine with haem and with each other to form haemoglobin. That this is true is supported by the finding of only minute traces of free globin in the normal red cell (Winterhalter et al, 1969).

Total a and fl Chain Synthesis in Sideroblastic Anaemia Reticulocytes In the 10 patients with sideroblastic anaemia the ratio of total counts in the a-chains to

total counts in the fl chains (a :/? ratio) isolated from whole-cell precipitated globin was always less than unity, varying from 0.52 to 0.87 (Table IV). The a : p ratio in a single patient was

TABLE IV. Incorporation of [3H]leucine into a- and chains of sideroblastic anaemia reticulocytes (whole-cell precipitation)

Case

I 2

3 4 5 6 7 8 9

11* I 0

-

Type of'anaemia

Hereditary Hereditary Hereditary Hereditary Idiopathic acquired Idiopathic acquired Idiopathic acquired Idiopathic acquired Idiopathic acquired Idiopathic acquired Pyridoxine-

responsive

Total counts ( C P )

a

I5990 31872 I 2400 14790 10400 14830 I9474 21126 12233 10612 27880

- Ratio a:b

0.66 0.78 0.52

0.75 0.81 0.87 0.70 0.72 0.79 0.81 I .07

- * Patient in remission at time of study.

always constant when measured in duplicate experiments on the same day, but did vary (& 0.1) when measured over several weeks. No correlation was found between the severity of the anaemia, percentage of hypochromic cells, Hb A2 level and the a:/? ratio. It is of interest that the a:/? ratio of the patient with the pyridoxine-responsive anaemia (Case 11),

who was in remission at the time of study, was between 1.0 and 1.07 on four separate estima- tions and the Hb A, was also found to be normal.

The a and p chains in the above experiments were isolated from globin precipitated from whole cells, a procedure which results in all the peptide chains in the cell being recovered, separated and the counts measured (see Methods). The finding of a low a :/? ratio, therefore, implies that there is a net deficiency in the synthesis ofa chains in the sideroblastic reticulocyte.

In eight patients with sideroblastic anaemia purification of the haernoglobin on Sephadex CM-50 prior to isolation of the globin and separation of the chains resulted in a marked decrease in the counts in both chains (Table V). Reduction in counts recovered from the

Globin Synthesis in Sideroblastic Anaemia 269

globin of haemoglobin compared to whole-cell globin ranged from 19 to 46% in the various patients. In any individual patient, the loss of counts resulting from haemoglobin purification varied by & 5% when measured on separate occasions. It is probable, therefore, that in the sideroblastic reticulocyte a large proportion of the peptide chains synthesized are not associa- ted with haemoglobin but are present as free chains. Attempts to recover these chains off the Sephadex CM-50 column by the addition of haem to the eluting buffer after the haemoglobin had come off were not successful. However, using Sephadex G-IOO chromatography, these free chains were detected as a peak of radioactivity eluting behind the main haemoglobin peak from the lysates of two patients (Fig 1.). The molecular weight of the protein in this peak was calculated to be approximately 34 000. After eluting it off the column it was concen- trated by ultra-filtration at 4°C for 24 hr, and the globin was precipitated with acid acetone

Case

-- I

5 6

7 8

2

9

I0

TABLE V. Incorporation of [3H]leucine into a and B chains of sideroblastic anaemia reticulo- cyres isolated from whole-cell precipitates and purified haemoglobin

~

Whole-cell ppt; total counts

( C P ) Ratio a : p

0: B

17750 21736 0.81

26000 32500 0.8 62916 71163 0.88

10871 13588 0.75 21126 29022 0.72

33200 44250 0.82

12333 16081 0.79

10612 12941 0.81

12986 25614 20540 50332 9249 7500 13732 8383

Purijied Hb; total counts

(cpm)

16324 30134 27625 56455 11899 9647 18283 10482

Decrease in counts (%I Ratio

36 I 33 1 0.79 39 I9 20

25 31 35 21

46 IS I9 26 29 37 I9

0.85 0.74 0.89 0.77 0.79 0.75 0.71

after the addition of 40 mg of unlabelled Hb A as a carrier. Following the separation of the a and p chains the radioactivity was found to be associated with both the a chain and the /3 chain peaks, with slightly more total counts (5-10%) in the /l chain. These findings confirm that in the sideroblastic reticulocyte the loss of radioactivity on purification of the haemo- globin is in all likelihood due to free a and /? chains which exist in the cell as ap dimers not associated with haem.

The ratio of total counts in the a chain to total counts in the p chain isolated from purified haemoglobin was between 0.71 and 0.89 in all the anaemic sideroblastic patients studied, thus strongly supporting the concept that in these reticulocytes there is a deficiency, albeit slight, in the synthesis of the a chain.

The E$ect ofHaem on a and p Chain Synthesis in Sideroblastic Anaemia Reticulocytes The detection of a large proportion of haem-depleted free a/3 dimers in the sideroblastic

reticulocyte is evidence that there is a deficiency of haem within these cells. Experiments were therefore carried out to study the effect of exogenous haem on the synthesis of the globin chains. The addition of haem at a concentration of 10- molesll to the incubation

270 J. M. White, M. C. Brain and M. A. M. Ali

mixture markedly increased the total counts in both chains between IOO and 200% in six patients studied (Table VI), whether the chains were isolated from the whole-cell precipitates (Table VI), or from purified haemoglobin (Table VII). Part of the increase in counts in both

- ( 0 )

Blue dextran - -

0.8 -

- 0.6 -

E

20

18

16

14

-12 N 0

Fraction no.

0 BD

0 0

0.4

0.2

Fraction no.

FIG I. G r o o gel filtration of lysate of [3H]leucine labelled sideroblastic cells: (a) cells lysed without haem; 0, OD 280 nm; x , cpm. (b) cells lysed in the presence of haem.

0 - 8

-6

-4

-2

-

-

I r--r-x+x I I

chains could be due to the combination of the free chains with haem and their conversion into haemoglobin. However, the increase in the counts in the chains isolated from the whole- cell must mean that synthesis of the chains had been stimulated. The addition of haem was also found to reduce the amount of ct and p chains which were retained by Sephadex CM-so

Giobin Synthesis in Sideroblastic Anaemia 271

chromatography in the purification of haemoglobin to less than 5% (Table VII), indicating that the free a and /? chains previously detected were haem-deficient and that they were capable of combining with haem to form haemoglobin. This was confirmed by Sephadex GIOO chromatography of a lysate to which haem had been added (Fig Ib), when the radio- active a/? dimer peak previously detected was no longer present. Another important finding was that the increase in counts in the a chain was always greater than that in the /? chain (Table VI). It is felt, however, that the data so far available are insufficient for their significance to be assessed.

Case

TABLE VI. Effect of haem on the total [3H]leucine incorporated into a and B chains of sidero- blastic anaemia reticulocytes isolated from whole-cell precipitates

~

Without haem; total counts

1 C P )

Increase in counts (%I

a l p

Ratio a:j3

B

I803 3 27707 14129 30590 34458 19283

a -~ 28715 ,50836 27923 70540 57636 55937

-

Ratio a:j3

B

34947 60008 28871 86210 71329 70252

0.76 0.78 0.83

0.77 0.82 0.76

-

I 2

3 7 8 9

~~

W i t h haem; total counts

(CP4

13872 21151

11764 23718 29132 14716

I I I

0.82 0.85 0.97 0.81 0.8 0.79

Whole-cell ppt; total counts

Case ( C P 4

U B ~~

I 47361 56933

7 61923 74308 9 57080 75433

2 78007 101419

TABLE VII. Effect of haem on total counts in a and B chains of sideroblastic anaemia reticulo- cytes isolated from whole-cell precipitates and purified haemoglobin

Purified Hb; total counts

Ratio (CP4 a:B

a B

0.83 45466 54086

0.82 59136 72077 0.75 55938 71851

0.76 77227 96350

Decrease in counts 1%)

a B --- 4 5 I 5 4 3

Ratio a:B

0.84 0.80 0.82

Case

1 2

7 9

Whole-cell ppt; total counts

(CPm)

U B

47361 56933

61923 74308 57080 75433

78007 101419

I I I I I

Ratio a:B

0.83 0.76 0.82 0.75

Ratio a:B

Purified Hb; total counts

(CP4

a B

45466 54086 77227 94350 59136 72077 55938 71851

a -- 4

4 I

0.84 0.80 0.82

B

5 5 3

It should be added that experiments were also carried out to study the effect of haem at a concentration of 10- 'M on the synthesis of the a and /? chains of control non-sideroblastic patients. In two patients there was a stimulation of both chains to a level of 10-15% above non-haem tubes, but in another two there was a significant depression to the order of 30%. The interpretation of these findings is not clear at the present time and must await further evaluation.

272 J. M. White, M. C. Brain and M. A. M. Ali

DISCUSSION

Defective synthesis of haem relative to globin by bone marrow erythroblasts of patients with sideroblastic anaemia has been reported previously (Bower et al, 1967; Waltuch et al, 1968; Kramer et al, 1969). No defect in globin synthesis was observed by Waltuch et al (1968). Heller et al (1965)~ however, found that the in vivo incorporation of ['4C]glycine into globin was less in a patient with sideroblastic anaemia than in normal subjects.

In the experiments reported in this paper the defective synthesis of globin by reticulocytes ofpatients with sideroblastic anaemia is revealed. It has been further shown that the synthesis of globin by these reticulocytes is abnormal in two respects; first, a large proportion of the peptide chains synthesized cannot be recovered as haemoglobin but are detectable as free

dimers, and, secondly, that the synthesis of the a and B peptide chains is asynchronous with an apparent deficiency in cc chain synthesis. The first of these defects in globin synthesis can be corrected by the addition of exogenous haem to the reticulocytes in vitvo, and the second defect may also be partially corrected by the haem addition.

Our findings must be interpreted with caution as with all experiments concerning haemo- globin synthesis by reticulocytes. Nevertheless they would appear to provide further evidence as to the possible nature of the defect in haemoglobin synthesis in sideroblastic anaemia.

Haenioglobin synthesis by reticulocytes of normal human subjects and animals is a regu- lated process, haem and globin being synthesized in equal proportions (Kruh& Borsook, 1956) , with the result that very little excess of either can be detected in the mature red cell (W' inter- halter et al, 1969). Our own findings ofct and B chain synthesis and assembly in the non-haem deficient control reticulocytes also indicate that there is very little free globin within the normal cell, in that over 95% of the labelled a and /3 chains could be recovered from the pure haemoglobin fraction of the cell. We are unable from our experiments to provide evidence as to the mechanism of this for our incubation times were too long. The findings indicate, however, that if between chain assembly and haemoglobin formation there is a free chain compartment within the cell then the transition time is probably rapid, otherwise a substantial pool of labelled free chain should be detectable even at 60 min and this appears not to be the case.

The main control is thought to be the synthesis of haem. Haem has been shown to control its own synthesis by end product inhibition (Karibian & London, 1965) or end production repression of 6 amino leavulinic acid synthetase, the rate limiting step in the haem synthetic pathway (Granick & Kappas, 1967; Kappas & Granick, 1968).

The effect of haem on globin synthesis is more complex. Haem has been shown to stiniu- late globin synthesis in the reticulocyte and bone marrow erythroblast (Bruns & London, 1965; Grayzel et al, 1966; Waxman & Rabinovitz, 1966; Waxman et al, 1967; Waxman, 1970) and in cell-free systems (Zucker & Schulman, 1968; Adamson et al, 1968, 1969). The influence of haem would appear to be at two stages of globin formation. Haem has been shown to stabilize polysomes on messenger RNA (Waxman & Rabinovitz, 1966; Grayzel et al, 1966; Zucker & Schulman, 1968) thus influencing the number of protein synthesizing units (Hunt et al, 1969). Haem also influences the assembly of the peptide chains synthesized (Tavill et al, 1968). Tavill et a1 (1968) found in reticulocytes of iron deficient rabbits that although the ratio of a to B chains synthesized was normal fewer a chains were incorporated

Globin Synthesis in Sideroblasfic Anaemia 273 into haemoglobin, that there was a large pool of a chains present both as monomers and as ap dimers, and that the addition of haem stimulated the formation of haemoglobin by co- ordinating c1 and p chain assembly and converting the ap dimer to the normal tetramer.

The studies of globin synthesis by reticulocytes of patients with sideroblastic anaemia reported in this paper both resemble and differ from the observations of Tavill et a1 (1968) in iron deficient rabbit reticulocytes. A pool ofap dimers was found, which like the observations of Tavill and co-workers in iron deficient rabbits was diminished by the addition of haem. However, whereas in the rabbit the synthesis of ct and /3 chains was equal, in sideroblastic anaemia reticulocytes there was a significant reduction in the radioactivity incorporated into c1 chains when measured in the whole cell lysate. The explanation for the diminished a chain synthesis in our experiments is not apparent. It may reflect a species difference or a difference between the disorder in haem synthesis due to iron deficiency and in sideroblastic anaemia. A possible explanation for the diminished a chain synthesis may be provided by the observation of Blum et al(1967,1970) that the addition of haem-depleted a or p peptide chains specifically inhibits the synthesis of homologous peptide chain in a cell free system in uitro, but that the addition of 01 chain stimulated #I chain synthesis but not the converse. Thus the presence of an a chain pool will not only result in an increase in the synthesis of /3 chains relative to tl chains, but cause inhibition of a chain production. In support of our data, it is of interest that a diminished synthesis of a chains relative to p chains has also been found in iron deficient patients (J. M. White and A. V. Hoffbrand, unpublished observations; D. J. Weatherall, personal communication) and also in lead poisoning (J. M. White, unpublished observations). These two conditions one assumes are associated with a deficiency of haem within the cell.

The finding of a low Hb A2 level in the blood of patients with sideroblastic anaemia (Reed & Mollin, 1968) and our further observation that it is lowest in the hypochromic cells might reflect a deficiency in ct chains available to combine with 6 chains. However, we have not studied 6 chain synthesis in sideroblastic reticulocytes, nor has the effect of iron deficiency on 6 chain synthesis been studied as far as we are aware. If the low Hb A, is due to the lack of a chains available to combine with 6 chains, the findings in sideroblastic anaemia would be analogous to a thalassaemia.

The cause of the haem deficiency in the reticulocytes cannot be deduced from the experi- ments presented here, but evidence localizing the defect will be presented in a subsequent communication. However, defects in the haem synthetic pathway in sideroblastic anaemia have been shown or inferred (Vavra & PoK 1967; Vogler et al, 1968; Rothstein et al, 1969).

All the patients with sideroblastic anaemia studied, although a heterogeneous group, showed similar haematological features; abnormal accumulation of iron in developing ery- throblasts and hypochromia of a proportion of circulating red cells. These features alone are evidence of disordered haemoglobin synthesis. The finding of a large amount of haem- depleted peptide chains within the reticulocytes provides direct biochemical evidence for the disorder in haemoglobin formation. Of more importance is the finding that the globin pre- pared from the whole cell consisted of nearly equal amounts of peptides chains as c1 and /3 dimers as in the form of haemoglobin tetramer. Thus the abnormality in haemoglobin syn- thesis may be as much due to failure of haemoglobin formation from monomer and dimer as to deficient peptide synthesis. That the failure of haemoglobin formation is secondary to haem deficiency is supported by the finding that the addition of exogenous haem to the reticulocytes

,

274 J. M. White, M. C. Brain and M. A. M. Ali

in vitro not only stimulated globin synthesis but also combined with free globin within the cell to form haemoglobin.

ACKNOWLEDGMENTS

We thank Mr A. S. Malik for his highly skilled technical assistance and Professor J. V. Dacie for his encouragement and support throughout this study.

REFERENCES

ADAMSON, S.D., HWERT, E. & GODCHAUX, W., 111 (1968) Factors affecting the rate of protein synthesis in lysate systems from reticulocytes. Archives of Biochemistry and Biophysics, 125, 671.

ADAMSON, S.D., HERBUT, E. & KJMP, S.F. (1969) Effects of hemin and other porphyrins on protein synthesis in a reticulocyte lysate cell-free system.

Journal of Molecular Biology, 42, 247. BANK, A. & MARKS, P.A. (1966) Excess of a chain

synthesis relative to B chain synthesis in thalassemia major and minor. Nature, 212, 1198.

BANK, A. & MARKS, P.A. (1969) Genetic control of hemoglobin synthesis and the thalassemia syndro- mes. Medical Clinics OfNorth America, 53, 875.

BARGELLESI, A., PONTREMOLI, S., MENIM, C. & CONCONI, F. (1968) Excess of a-globin synthesis in homozygous fi-thalassemia and its removal from the red blood cell cytoplasm. European Journal of Biochemistry, 3 , 364.

BLUM, N., MALEKNIA, M. & SCHAPIRA, G. (1970) a- et pglobines libres et biosynthese de I'hemoglo- bine. Biochimica et Biophysica Acta, 199, 236.

BLUM, N. & SCHAPIRA, G. (1967) Regulation de la synthese de I'himoglobine par addition de a- hkmoglobine libre. Comptes Rendus Hebdomadaire de Sbances de l'dcadthie des Sciences, D, 264, 1211.

BOUSSER, J., GAJDOS, A., GAJDOS-TBROK, M., BILSKI- PASQUIER, G. & ZITTOUN, R. (1967) AnCmie sidero- blastique idiopathique acquise: incorporation de la glycine-2-c-14 dans l'hhme et la globine des Crythroblastes midullaires in vitro. Nouvelle Revue Franpise d'Ht!matologie, 7, 847.

BRAY, G.A. (1960) A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintilla- tion counter. Analytical Biochemistry, I, 279.

BRUNS, G.P. & LONDON, LM. (1965) The effect of hemin on the synthesis of globin. Biochemical and Biophysical Research Communications, 18, 236.

CLEGG, J.B., NAUGHTON, M.A. & WEATHERALL, D.J. (1966) Abnormal human haemoglobin. Separation and characterization of the a and fi chains by chromatography and the determination of two new variants, Hb Chesapeake and Hb J (Bangkok).

Journal of Molecular Biology, 19, 91.

DACIB, J.V. &LEWIS, S.M. (1968) Practical Haematology, 4th edn. Churchill, London.

DANON, D., MARIKOVSKY, Y. & GASKO, 0. (1966) 51Chromium uptake as a function of red cell age.

Journal of Laboratory and Clinical Medicine, 67, 70. GRANICK, S. & KAPPAS, A. (1967) Steroid induction of

porphyrin synthesis in liver cell culture. I. Struc- tural basis and possible physioIogica1 role in the control of heme formation. Journal of Biological Chemistry, 24,4587.

GRAYZEL, A.I., H~RCHNER, P. & LONDON, I.M. (1966) The stimulation of globin synthesis by heme. Proceedings of the National Academy of Sciences of the United States of America, 55, 650.

HEILMEYER, L. (1967) In-vitro-Testung Bssensibler Mimien. Blut, 16, I.

HELLER, P., STONE, J.V., APPLE, D. & COLEMAN, R.D. (1965) Defective globin synthesis in hypochromic hypersideremic anemia (m-thalassemia?). Blood, 25,

HUNT, T., HUNTER, T. & MUNRO, A. (1969) Control ofhaemoglobin synthesis: Rate of translation of the messenger RNA for the a and /3 chain. Journal of Molecular Biology, 43, 123.

KAPPAS, A. & GRANICK, S. (1968) Steroid induction of porphyrin synthesis in liver cell culture. 11. The effects of heme, uridine diphosphate glucuronic acid, and inhibitors of nucleic acid and protein synthesis on the induction process. Journal of Biological Chemistry, 243, 346.

KARIBIAN, D. & LONDON, LM. (1965) Control of heme synthesis by feedback inhibition. Biochemical and Biophysical Research Communications, 18, 243.

KRAMER, S., VILJOEN, E., BECKER, D., ZAIL, S.S. & METZ, J. (1969) The relationship between haem and globin synthesis by erythroid precursors in refrac- tory normoblastic anaemia. Scandinavian Journal of Haematology, 6, 293.

KRUH, J. & BORSOOK, H. (1956) Haemoglobin syn- thesis in rabbit reticulocytes in vitro. Journal of Biological Chemistry, 220, 905.

LINGREL, J.B. & BORSOOK, H. (1963) A comparison of amino acid incorporation into the hemoglobin and ribosomes of marrow erythroid cells and circulating

635.

Globin Synthesis in Sideroblastic Anaemia 275 reticuloojtes of severely anemic rabbits. Biochemis- try, 2, 309.

MARENGO-ROWE, A.J. (1965) Rapid electrophoresis and quantitation of haemogiobins on cellulose acetate. Journal of Clinical Pathology, 18, 790.

MODELL, C.B., LATTER, A., STEADMAN, J.H. &HUEHNS, E.R. (1969) Haemoglobin synthesis in &thalassae mia. British Journal ofHuemutology, 17, 485.

MOLLIN, D.L. & HOFFBRAND, A.V. (1968) Siderobfas- tic anaemia. Recent Advances in Clinical Putholegy, Series V, p 273. Churchill, London.

REED, L.J. & MOLLIN, D. (1968) Synthesis of haemo- globin Az in sideroblastic and megaloblastic anae- mias. (Abstract). Abstracts XIICongr. Int. SOC. Hemat., 65.

ROTHSTEIN, G., LEE, G.R. & CARTWRIGHT, G.E. (1968) Refractory sideroblastic anaemia associated with reduced heme synthetase activity in the reticdo- cytes. (Abstract). AbstractsXIICongr. Int. SOC. Hemat., 65.

ROTHSTEIN, G., LEE, G.R. & CARTWRIGHT, G.E. (1969) Sideroblastic anemia with dermal photosensitivity and greatly increased erythrocyte protoporphyrin. N e w England Journal of Medicine, 280, 587.

TAVILL,A.S., GRAYZEL, A.I., LONDON, I.M., WILLIAMS, M.K. & VANDERHOFP, G.A. (1968) The role of heme in the synthesis and assembly of hemoglobin. Journal of Biologicat Chemistry, 243, 4987.

VAVRA, J.D. & POFP, S.A. (1967) Heme and porphyrin synthesis in sideroblastic anemia. Journal of labora- tory and Clinical Medicine, @, 904.

VOGLER, W.R. & MINGIOLI, E.S. (1965) Heme syn- thesis in pyridoxine-responsive anemia. New Eng- land Journal ofMedicine, 273, 347.

WAXMAN, H.S. (1970) Stimulation of globin synthesis;

relative responsiveness of reticulocytes and nuclea- ted erythroid cells. Journal of Clinical Investigation, 49, 701.

WAXMAN, H.S., FREEDMAN, M.L. & RABINOVITZ, M. (1967) Studies with s9Fe-labeled hemin on the control of polyribosome formation in rabbit reticulocytes. Biechimica et Biophysica Actu, 145, 3 5 3 .

WAXMAN, H.S. & RABINOVITZ, M. (1965) Iron supple- ment in vitro and the state of aggregation and function of reticulocyte ribosomes in hemoglobin synthesis. Biochemical and Biophysical Research Communications, 19. 538.

WAXMAN, H.S. & RABINOVITZ, M. (1966) Control of retidocyte polyribosome content and hemoglobin synthesis by heme. Biochimicb et Biophysicu Acta, 129, 369.

WHITE, J.M. &BRAIN, M.C. (1970) Defective synthesis of an unstable haemoglobin: Haemoglobin Koln (DQ8 Val-Met). British Journal of Haematology, 18, 195.

WHITE, J.M., BRAIN, M.C. & ALI, M.A.M. (1969) a- and /%peptide chain synthesis in sideroblastic anaemia. (Abstract). British Journal of Huematology,

WINTERHALTER, K.H., HEYWOOD, J.D. HUEHNS, E.R. & FINCH, C.A. (1969) The free globin in human erythrocytes. I. British Journal of Huematology, 16,

WALTUCH, G., LANZEROTTI, A.K. & SCHRIER, S.L. (1968) Marrow defect in idiopathic ineffective erythropoiesis. Annuls of Infernal Medicine, 68, 1005.

ZUCKBR, W.V. & SCHULMAN, H.M. (1968) Stimula- tion of globin-chain initiation by haemin in the reticulocyte cell-free system. Proceeding ofthe Nution- a1 Academy ofsciences of the United States of America, 59, 582.

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