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ERYTHROPOIESIS-INHIBITING FACTOR IN URINE
781
ERYTHROPOIESIS-INHIBITING FACTOR IN URINE
SIR,-In the past few years the existence of an inhibitor of erythropoiesis in various conditions has been suggested by several research-workers.1-6 Lewis et awl. recently described a predominently basic protein, with a potent inhibitory effect
on
erythropoietin,
in the urine of normal
healthy
individuals. Their paper prompts us to present a preliminary report of our data which agree with their findings. The demonstration of this inhibitor suggests that an activatorinhibitor complex may play a part in the normal regulation of erythropoiesis. Urines from normal healthy controls, from a patient with aplastic ansemia, and from patients with severe anxmia of chronic renal failure were collected for 24-hour periods and immediately frozen at -20°C after each voiding. The following procedures were performed at 4°C: the urines were dialysed and concentrated using ’Carbowax’; and after centrifugation the supernatants were lyophilised and stored for further investigation. Part of the urine-concentrate was passed through a ’ Sephadex G 100 ’ column (1-6 x 110 cm.), eluted with a 0-05 M tris-hydrochloride buffer (pH 7-4) containing 0-1 M potassium chloride. Approximately 3 ml. of the effluents were collected in each tube. The concentration of the proteins was determined spectrophotometrically at 280 m[L. All fractions were tested for stimulation and inhibition of erythropoiesis by a slight modification of our technique using mice made polycythxmic by hypoxia(which measures Fe 59 incorporation into red-cells). The inhibitory effect of the proteins was tested by incubating 0-5 mg. of the material for 60 minutes at 37°C with our laboratory standard erythropoietin before injection into the mice. In the urine from all three groups, a fraction with a
relatively
small molecular
weight,
as
determined
on
the
Erslev, A. J., Kazal, L. A. Proc. Soc. exp. Biol. Med. 1968, 128, 845. Fisher, J. W., Hatch, F. E., Roh, B. L., Allen, R. C., Kelly, B. J. Blood, 1968, 31, 440. 3. Krzymowski, T., Krzymowska, H. ibid. 1962, 19, 38. 4. Kuratowska, Z. Ann. N. Y. Acad. Sci. 1968, 149, 128. 5. Lewis, J. P., Neal, W. A., Moores, R. R., Gardner, E. Jr., Alford, D. A., Smith, L. L., Wright, C. S., Welch, E. T. J. Lab. clin. Med. 1969, 74, 608. 6. Whitcomb, W. H., Moore, M. Z. ibid. 1965, 66, 641. 7. Lindemann, R., Trygstad, O., Halvorsen, S. Scand. J. Hœmat. 1969 6, 77. 1. 2.
sephadex column, was found to have an inhibitory effect erythropoietin. The results are shown in the accompanying figure. Varying doses of our standard erythropoietin (E.S.F.) were incubated with 0-5 mg. of the inhibitor protein, and the results plotted against the dose-response curve of the standard E.S.F. The strongest inhibitory effect was found in urine from normal subjects and from patients with aplastic ansemia. This inhibitory fraction was well separated from a fraction with an active erythropoiesis-stimulating effect in both urines. The findings of a relatively slight inhibitory effect, as well as no active erythropoietin excretion in 24hour urines from 4 patients with chronic renal failure, suggests that the site of production of both the activator (E.s.F.) and the inhibitor (E.I.F.) may be the kidney. Other sites of production cannot, however, be excluded. Lewis et al. found that the inhibitor protein was predominantly basic, which could explain the interaction with the acidic glycoprotein of erythropoietin. On the basis of their findings, we conclude that an erythropoiesis-inhibiting factor, as well as an erythropoiesisstimulating factor, may play a central part in the normal regulation of erythropoiesis, and that variations in amounts of the different factors could explain some types of anaemia or polycythxmia. on
Children’s Department,
Rikshospitalit, Oslo, Norway.
ROLF LINDEMANN.
DIAGNOSIS OF COLOUR-VISION DEFECTS IN VERY YOUNG CHILDREN interested in the letter from Dr. Sassoon and was SIR,-I Dr. Wise on this subject (Feb. 21, p. 419). For the reasons they give, testing of colour vision in very young children is important. Some years ago I cooperated in research to determine how early the Farnsworth ’100-hue ’ test could usefully be employed. We began testing children at the age of eleven, and then at progressively younger ages down to school beginners at age five. Down to about the age of eight the answers were satisfactory, but below that the errors were extreme. It seemed that the nsvcholosical conception of colour order by wavelength has to be learned like any other conception. Normally one supposes that this is learned by noticing the arrangement of the Dr. colours in the rainbow. Sassoon and Dr. Wise’s technique with the ’D-15 ’ test is slightly different, in that at each individual cap the child is asked to choose the nearest colour, and not as in the 100-hue test where a choice is made freely from (at the beginning) twenty-one caps at a time. For their purpose this seems simpler. Terman
and
Merrill, in the
1940s, found, when working
Fe" uptake in red-cells of polycythaemic mice after injection with different doses of erythropoietin (E.S.F.) incubated with inhibitor protein.
normal urine, = urine from patient with aplastic with chronic renal failure. The line represents response to erythropoietin alone. =
anasmia; A
=
urine from patients
on
their intelligence test, that up to the age of three colour was of less significance than shape, and that choice was made by shape rather than hue, so it may be that colour vision is a relatively late development. Japanese children use a brush in their reading and writing, and so are accustomed to following patterns. The Ishihara test incorporates some non-numerate patterns, and I have tried with very young children to get them to
ERYTHROPOIESIS-INHIBITING FACTOR IN URINE
SIR,-In the past few years the existence of an inhibitor of erythropoiesis in various conditions has been suggested by several research-workers.1-6 Lewis et awl. recently described a predominently basic protein, with a potent inhibitory effect
on
erythropoietin,
in the urine of normal
healthy
individuals. Their paper prompts us to present a preliminary report of our data which agree with their findings. The demonstration of this inhibitor suggests that an activatorinhibitor complex may play a part in the normal regulation of erythropoiesis. Urines from normal healthy controls, from a patient with aplastic ansemia, and from patients with severe anxmia of chronic renal failure were collected for 24-hour periods and immediately frozen at -20°C after each voiding. The following procedures were performed at 4°C: the urines were dialysed and concentrated using ’Carbowax’; and after centrifugation the supernatants were lyophilised and stored for further investigation. Part of the urine-concentrate was passed through a ’ Sephadex G 100 ’ column (1-6 x 110 cm.), eluted with a 0-05 M tris-hydrochloride buffer (pH 7-4) containing 0-1 M potassium chloride. Approximately 3 ml. of the effluents were collected in each tube. The concentration of the proteins was determined spectrophotometrically at 280 m[L. All fractions were tested for stimulation and inhibition of erythropoiesis by a slight modification of our technique using mice made polycythxmic by hypoxia(which measures Fe 59 incorporation into red-cells). The inhibitory effect of the proteins was tested by incubating 0-5 mg. of the material for 60 minutes at 37°C with our laboratory standard erythropoietin before injection into the mice. In the urine from all three groups, a fraction with a
relatively
small molecular
weight,
as
determined
on
the
Erslev, A. J., Kazal, L. A. Proc. Soc. exp. Biol. Med. 1968, 128, 845. Fisher, J. W., Hatch, F. E., Roh, B. L., Allen, R. C., Kelly, B. J. Blood, 1968, 31, 440. 3. Krzymowski, T., Krzymowska, H. ibid. 1962, 19, 38. 4. Kuratowska, Z. Ann. N. Y. Acad. Sci. 1968, 149, 128. 5. Lewis, J. P., Neal, W. A., Moores, R. R., Gardner, E. Jr., Alford, D. A., Smith, L. L., Wright, C. S., Welch, E. T. J. Lab. clin. Med. 1969, 74, 608. 6. Whitcomb, W. H., Moore, M. Z. ibid. 1965, 66, 641. 7. Lindemann, R., Trygstad, O., Halvorsen, S. Scand. J. Hœmat. 1969 6, 77. 1. 2.
sephadex column, was found to have an inhibitory effect erythropoietin. The results are shown in the accompanying figure. Varying doses of our standard erythropoietin (E.S.F.) were incubated with 0-5 mg. of the inhibitor protein, and the results plotted against the dose-response curve of the standard E.S.F. The strongest inhibitory effect was found in urine from normal subjects and from patients with aplastic ansemia. This inhibitory fraction was well separated from a fraction with an active erythropoiesis-stimulating effect in both urines. The findings of a relatively slight inhibitory effect, as well as no active erythropoietin excretion in 24hour urines from 4 patients with chronic renal failure, suggests that the site of production of both the activator (E.s.F.) and the inhibitor (E.I.F.) may be the kidney. Other sites of production cannot, however, be excluded. Lewis et al. found that the inhibitor protein was predominantly basic, which could explain the interaction with the acidic glycoprotein of erythropoietin. On the basis of their findings, we conclude that an erythropoiesis-inhibiting factor, as well as an erythropoiesisstimulating factor, may play a central part in the normal regulation of erythropoiesis, and that variations in amounts of the different factors could explain some types of anaemia or polycythxmia. on
Children’s Department,
Rikshospitalit, Oslo, Norway.
ROLF LINDEMANN.
DIAGNOSIS OF COLOUR-VISION DEFECTS IN VERY YOUNG CHILDREN interested in the letter from Dr. Sassoon and was SIR,-I Dr. Wise on this subject (Feb. 21, p. 419). For the reasons they give, testing of colour vision in very young children is important. Some years ago I cooperated in research to determine how early the Farnsworth ’100-hue ’ test could usefully be employed. We began testing children at the age of eleven, and then at progressively younger ages down to school beginners at age five. Down to about the age of eight the answers were satisfactory, but below that the errors were extreme. It seemed that the nsvcholosical conception of colour order by wavelength has to be learned like any other conception. Normally one supposes that this is learned by noticing the arrangement of the Dr. colours in the rainbow. Sassoon and Dr. Wise’s technique with the ’D-15 ’ test is slightly different, in that at each individual cap the child is asked to choose the nearest colour, and not as in the 100-hue test where a choice is made freely from (at the beginning) twenty-one caps at a time. For their purpose this seems simpler. Terman
and
Merrill, in the
1940s, found, when working
Fe" uptake in red-cells of polycythaemic mice after injection with different doses of erythropoietin (E.S.F.) incubated with inhibitor protein.
normal urine, = urine from patient with aplastic with chronic renal failure. The line represents response to erythropoietin alone. =
anasmia; A
=
urine from patients
on
their intelligence test, that up to the age of three colour was of less significance than shape, and that choice was made by shape rather than hue, so it may be that colour vision is a relatively late development. Japanese children use a brush in their reading and writing, and so are accustomed to following patterns. The Ishihara test incorporates some non-numerate patterns, and I have tried with very young children to get them to