1048 AN ERYTHROCYTE MEMBRANE-PROTEIN ANOMALY IN MARCH HÆMOGLOBINURIA

J. C. FINDER J. P. BANGA D. C. LINCH W. B. GRATZER E. R. HUEHNS Department of Immunology, Middlesex Hospital, London; M.R.C. Biophysics Unit, King’s College London; and Department of Clinical Hæmatology, University College Hospital Medical School, London In three patients with march hæmoglobinuria a well-defined protein abnormality has been revealed by high-resolution sodium-dode-

Summary

cyl-sulphate/polyacrylamide-gel electrophoresis. Introduction IN march haemoglobinuria hxmoglobin appears in the urine after prolonged marching or running on hard roads’ and after excessive conga drumming2 or karate.3 It is due to traumatic intravascular haemolysis in the feet or hands. The cause of this form of haemoglobinuria could be either environmental stress or an intrinsic (inherited) red-cell abnormality. The first implies that any individual can develop this condition providing the appropriate form of mechanical stress is applied to the red cells in vivo, and there is some experimental evidence for this.4 Although the red cells have previously been studied in detail without revealing any consistent abnormality, newer methods of studying cell-membrane proteins were not used.

globin

was 16.7 g/dl both before and after exercise, and Coulter output and a blood film were normal.

Method

Red-cell ghosts were prepared from fresh heparinised blood a modification of the method of Dodge et al.5 A portion of the ghost pellet was dissolved in 20% (w/v) sodium dodecyl sulphate (SDS) and dialysed overnight against the buffer used to make the stacking gel. 2-mercaptoethanol was then added to give a final concentration of 5% (v/v), and the sample was immersed for 3 min in a bath of boiling water. Electrophoresis was done in a 5-15% (w/v) stacking gel, with the discontinuous buffer system of Laemmli.6 The gels were stained with Coomassie brilliant-blue. This gradient system affords high resolution and good reproducibility. 35 protein bands can be seen, including many of the lower-molecular-weight bands which cannot be satisfactorily resolved with other methods.7

by

Results The three patients studied show well-defined differfrom the normal pattern in the low-molecularweight region (see figure). These changes have not been seen in other cases of hsemolytic anaemia, including hereditary spherocytosis and hereditary elliptocytosis. The greatly diminished band in case 1 has an approximate molecular weight of 37 000 and the band present ences

Case-reports Case1 An 18-year-old man had a year-long history of passing red urine after road running. On these occasions he noticed a little discomfort in his feet but was otherwise well. On examination, no abnormality was detected. After a 4t mile run on a tarmac surface the urine became dark red and contained haemoglobin but not myoglobin or red cells. Plasma bilirubin was 22 mol/1 before exercise and afterwards rose to 33 umol/1. Plasma haptoglobin fell from 0.28 g/1 to 0-19 g/1 (normal range 0.3-1.9 g/1) The Coulter outputs (blood-counts on Coulter counter model S) before and after exercise were normal. A few spherocytes were seen on the blood film both before and after exercise. The reticulocyte count was 1-2%. Exercise on a bicycle did not produce hoemoglobinuria. Case 2

A 31-year-old man reported that he repeatedly passed red urine after running for 2t hours on the road. He had been running this distance for only a year and before this had never noticed red urine. There were no other symptoms, and no abnormality was detected on examination. The urine after exercise was dark red and contained h2emoglobin but no myoglobin or red cells. The post-exercise haemoglobin was 14.6 6 g/dl, the reticulocyte count was 0-7%, and the blood film was normal. Plasma bilirubin was 17 p.moVl. Liver-function tests, serum urea, and serum electrolytes were normal. Case 3

passed dark red urine on two occasions. 26-year-old episode followed a hard game of squash and the other apparently occurred after an excess of alcohol. There were no other symptoms at these times, and no abnormality was detected on examination. After a 2tmile road run, the urine contained hxmoglobin but not myoglobin or red cells. HasmoA One

man

Sodium-dodecyl-sulphate/polyacrylamide-gel electrophoresis patterns of normal erythrocyte membranes and membranes from patients with hereditary spherocytosis, hereditary elliptocytosis, and march haemoglobinuria. A, normal. B-E, hereditary spherocytosis. F, hereditary elliptocytosis. G, march hxmoglobinuria (case 1). H, march hxmoglobinuria (case 2). I, march hxmoglobinuria (case 3).z) J, actinomyosin consisting of myosin (MW 200 000) and actin ‘

I

(42 000). K, mixture of standard-molecular-weight proteins. The gel consists of a 5-15% (w/v) polyacrylamide gradient containing 0.375 mol/1 ’tris’ (pH 8-8) and 0-1% SDS. The cross-linker concentration was 2.5% of that of the total acrylamide. Initiator and catalyst were 0-035% ammonium persulphate and 0025 c TEMED. After the gel had set a stacking gel containing 3-75% acrylamide in 0.125 molll tris (pH 6-8) and 0-1% SDS was polymerised on top. Ten slots were formed and 25-30 ul samples containing approximately 40 ng protein were applied per channel.

1049 in normal

subjects

but absent in

our

three

patients

has

a molecular weight of 29 000.

Discussion is a

rare condition that affects young men, though it has been described in women.8 Although the passage of red urine may be the sole complaint, this symptom may be accompanied by nausea, abdominal pain, muscular aches, and a burning sensation in the feet.4 Jaundice may occur transiently. Anaemia is rare and mild if present, as the volume of blood haemolysed in an average paroxysm is only 6-40 ml.9 The reticulocyte count may be raised but is often normal. Morphological evidence of red-cell damage is not a usual feature. In the original description of march haemoglobinuria in 1881 Fleischer’ proposed that there was a primary hsematological disorder, but this view lost favour and in 1894 Dickinson’O suggested that it was a response to

March

haemoglobinuria

mainly

physiological stress. Davidson’s studies4 showed that individual running style, type of footwear, and running surface were all important; and, indeed, modification of any of these factors could bring an end to the episode. Our finding of an erythrocyte-membrane anomaly in three patients suggests that in these cases an intrinsic abnormality leads to increased susceptibility of the red cells to extreme mechanical trauma. There may therefore be two forms of march haemoglobinuria. One form may be a physiological phenomenon and the other may be due to red-cell anomalies such as extreme

Preliminary Communication FACTOR VIII-RELATED ANTIGEN (VIII R:AG) IN HÆMOPHILIC PATIENTS AND IN CARRIERS E.

DAVID N. FASS J. WALTER BOWIE Section of Hematology Research, Mayo Clinic and Mayo

Foundation, Rochester, Minnesota 55901, U.S.A. All of fourteen patients with severe classic hæmophilia and twelve of fifteen obligate carriers had a pre-peak ("rocket") above the sample well when factor VIII-related antigen was examined by crossed immunoelectrophoresis. The pre-peak may be found in other conditions-including von Willebrand’s disease—but if these conditions can be excluded, the pre-peak may be helpful in identification of carriers or

Summary

hæmophilia. INTRODUCTION

WHEN factor VIII-related antigen (VIII R:AG) is examined by crossed immunoelectrophoresis, some of the protein may remain at the origin and appear as a "rocket" (pre-peak) above the sample well. We noticed that the pre-peak is especially common in patients with classic haemophilia and report our preliminary observations in hsemophilic patients, obligate carriers of haemophilia, and normal people. PATIENTS AND METHODS

Blood was drawn into plastic syringes and anticoagulated BIith sodium citrate (1 volume of 3-8% sodium citrate to 9

have described here. The mechanism by which the demonstrated defect causes haemolysis is unclear, since the functions of the absent low-molecular-weight protein bands are not known. As this condition presents only after severe exercise, many people who have the red-cell anomaly will not be detected. This new marker will enable us to make appropriate family and population studies. We thank Prof. R. H. T. Edwards for referring two of the patients and Dr 1. Chanarin for providing blood samples from patients with hereditary spherocytosis. we

Requests for reprints should be addressed to E. R. H., Department Hoematology, University College Hospital Medical School, University Street, London WC1. of Clinical

REFERENCES 1. Fleischer R. Ueber eine neue Form von Hæmoglobinurie beim Menschen. Berl Klin Wschr 1881; 18: 691. 2. Kaden WS. Traumatic hæmoglobinuria in conga-drum players. Lancet 1970; i: 1341. 3. Streeton JA. Traumatic hæmoglobinuria caused by karate exercises. Lancet 1967; ii: 191. 4. Davidson RJL. March or exertional hæmoglobinuria. Semin Hæmatol 1969;

6: 150. 5.

Mitchell C. Hanahan DJ. The preparation and chemical characteristics of hæmoglobin-free ghosts of human erythrocytes. Arch Biochem

Dodge JT,

1963; 100: 119. 6. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680. 7. Banga JPS. The analysis of membrane protein components. PH.D. thesis, Council for National Academic Awards, 1979. 8. Gilligan DR, Altschule MD. March hæmoglobinuria in a woman. N Engl J Med 1950; 243: 944.

Gilligan DR, Blumgart HL. March hæmoglobinuria. Medicine (Baltimore) 1941; 20: 341. 10. Dickinson L. Hæmoglobinuria from muscular exertion. Trans Clin Soc Lond 9.

1894; 27: 230.

volumes of blood). Coagulant activity of factor VIII (VIII:C) was measured by the degree of correction of the partial thromboplastin time it produced in human haemophilia A plasma.’1 The antigenic activity associated with factor VIII (VIII R:AG) was detected by a rabbit antibody to purified Willebrand factor (Behring Diagnostics) and was measured by electroimmuno-

assay.2 Electrophoresis was carried out with a buffer consisting of 0-09 mol/! diethylbarbituric acid/sodium diethylbarbiturate and 0-01mol/1 disodium EDTA pH 8-6adjusted with sodium chloride to obain a specific conductance of 6 -1mQ-lcm-1 . Glass plates 13-1cmxll-1cm (J. M. Freed, Inc., Perkasie, Pennsylvania) were cleaned overnight in sulphuric acid dichromate and then washed 5 times in water. The plates were precoated with 0-2% agarose in water and dried overnight. The precoated plate was covered to a depth of 1 mm with 1% agarose dissolved in a 1/3 dilution of electrophoresis buffer. A ’Plexiglass’ template was used to ensure a uniformity of thickness. The solidified agarose gel was then removed except for two strips 1 cm in breadth running the length of the slide, one in the middle of the slide and one at the bottom. Wells 4 mm in diameter were cut at both ends of these two strips. A Drummond microdispenser (Drummond Scientific Co., Broomall, Pennsylvania) was used to place 10 ul of sample in each test well. Electrophoresis was carried out for 4 h at 4 V/cm at a temperature of 4°. The plate was placed so that the sample wells were at the cathodal end of each agarose strip and the strips, in turn, were connected to the electrophoresis reservoir by single thickness sheets of ’Telfa’ pad (Kendall, Chicago, Illinois). At the end of electrophoresis in the first dimension, antibody-containing agarose was poured onto the slide above the remaining agarose strips, again with the plastic template to uniform thickness of 1 mm. The agarose was melted in a 1/3 dilution of electrophoresis buffer and cooled to 50-540 before addition of a rabbit antihuman-factor VIII R:AG antibody (Behring Diagnostics) at a final dilution of 1:100. Elecensure a

_

An erythrocyte membrane-protein anomaly in march haemoglobinuria.

1048 AN ERYTHROCYTE MEMBRANE-PROTEIN ANOMALY IN MARCH HÆMOGLOBINURIA J. C. FINDER J. P. BANGA D. C. LINCH W. B. GRATZER E. R. HUEHNS Department...
368KB Sizes 0 Downloads 0 Views