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FACTOR-IX DEFICIENCY IN THE NEPHROTIC SYNDROME
1079
hypercholesterolxmia is related to excess circulating thyrotrophic hormone, a raised serum-cholesterol would be expected during the time that the failing thyroid was responding adequately to the increasing pituitary stimulus.
Methods
If the
analogy between prediabetes and premyxoedema possible aetiological factors in the development of coronary-artery disease. Thannhauser (1958) repeatedly warns against arguing from the particular to the general when considering the high incidence of atheroma in idiopathic familial hypercholesterolxmia. Nevertheless, factors affecting serum-cholesterol consistently influence the incidence of coronary-artery disease in the same direction. The following factors are associated There may be
an
as
with increased serum-cholesterol and increased incidence of coronary-artery disease: genetic predisposition, sedentary work, obesity, age, male sex, post-menopausal women (compared with premenopausal women), hypertension, idiopathic xanthomatosis, diabetes and prediabetes, and pre-myxoedema. Factors associated with decreased serumcholesterol and incidence of coronary-artery disease: include exercise and manual work, leanness, fasting and starvation, cestrogens, and thyrotoxicosis. There are many aetiological factors in coronary-artery disease. The importance of premyxoedema which might cause death from coronary-artery disease before the myxoedema itself develops can be assessed only by further studies. REFERENCES
Christie, J. F., Lyall, A., Anderson, T. E. (1930) Br. J. Derm. 42, 429. Craig, L. S., Lisser, H. (1944) J. clin. Endocr. Metab. 4, 12. Escamilla, R. (1942) ibid. 2, 33. Fowler, P. B. S., Briggs, J. H. (1962) Proc. R. Soc. Med. 55, 601. Gee, D. J., Goldstein, J., Gray, C. H., Fowler, J. F. (1959) Br. med. J. ii, 341. Sweitzer, S. E., Winer, L. H. (1940) Archs. Derm. Syph. 42, 419. Thannhauser, S. J. (1958) Lipidoses. New York and London. Thorp, J. M. (1962) Lancet, i, 1323. (1963) J. Atheroscler. Res. 3, 351. —
FACTOR-IX DEFICIENCY IN THE NEPHROTIC SYNDROME D. A. HANDLEY M.B. Adelaide, M.R.A.C.P., M.C.Path., M.C.P.A. HÆMATOLOGIST, INSTITUTE OF MEDICAL AND VETERINARY SCIENCE, ADELAIDE
J. R. LAWRENCE M.B.
DIRECTOR,
RENAL
Adelaide, M.R.A.C.P.
UNIT, QUEEN ELIZABETH HOSPITAL, WOODVILLE, SOUTH AUSTRALIA
Four patients with the nephrotic syndrome shown to have a deficiency of circulatfactor IX. In each, the abnormality was ing coagulation reversed when the nephrotic syndrome responded to steroid therapy.
Summary
were
Introduction
MANY
changes in the constituents of circulating blood
have been described in patients with the nephrotic
syndrome (Kark et al. 1958). In this paper we describe four nephrotic patients who were shown to have low levels of circulating coagulation factor ix during the acute phase of the illness which returned to normal with improvement in the nephrotic syndrome. The abnormality was observed for renal
during routine coagulation studies in preparation biopsy. The thromboplastin-generation screening test (Hicks-Pitney) gave abnormal results in each case, and subsequent assay of factor ix identified the deficiency.
The
bleeding-time (Duke), tourniquet test, whole-blood clotting-time (Lee and White), and one-stage " prothrombin "time (Quick) and fibrinogen assay were carried out as described by Biggs and Macfarlane (1962). The thromboplastingeneration screening test of Hicks and Pitney, as modified by MacPherson and Hardisty (1961), was used as a preliminary test of the plasma factors required for the intriasic thromboplastin system. Factor ix was assayed according to the method described by Biggs et al. (1961) which is based on the classical thromboplastin-generation test. Factor x was assayed according ing to the method of Denson (1961), using charcoal-filtered ox plasma as a source of factor-x-deficient substrate. Factor vm was assayed according to the method of Pitney (1956). Circulating anticoagulants affecting factor ix were tested by making mixtures of normal plasma and plasma from the patient. These mixtures were then tested in the Hicks-Pitney screeningtest system and the classical thromboplastin-generation test of Biggs and Douglas (1953). Urinary protein was assayed by the biuret method. Case-records Case 1 A 15-year-old boy was admitted to hospital with swelling of the legs and face. There was no preceding sore throat. He had gross oedema of the legs and face, and ascites. The bloodpressure was 115/65 mm. Hg. The results of investigations were:
titre,
blood-urea-nitrogen, 75 Todd
14 mg. per 100
units; throat culture,
no
ml.; antistreptolysin growth of pathogens.
Microscopic examination of the urinary sediment showed 10-15 hyaline and granular casts per high-power field; daily urinary protein excretion was 7-10 g.; serum-albumin, 0-51 g. per 100 ml.; serum-globulin, 3-1 g. per 100 ml.; serum-cholesterol, 720 mg. per 100 ml. For 2 months the patient was treated with diuretics without improvement in the nephrotic syndrome. He was then assessed for renal biopsy, but a defect was found in the thromboplastin-
generation screening-test. Assay of factor ix showed 25% activity (see table and fig. 1). Prednisolone 40 mg. daily was given and diuresis began on the 11th day. Proteinuria decreased sharply 18 days after steroid therapy was begun, and the serum-albumin rose to 3-0 g. per 100 ml. Subsequently the dose of prednisolone was reduced and then discontinued. There has been no recurrence of proteinuria after 2 years. Further assays of factor ix 6 and 7 months later showed 100% activity. The significance of the nephrotic syndrome in relation to factor-Ix deficiency was not at first appreciated in this patient, although extensive family studies showed no other evidence of Christmas disease. Case 2 A year after case 1 was admitted a farmer, aged 18 years, with acute nephritis which had come on 10 days after a severe sore throat, was admitted. His blood-pressure was 150/90 mm. Hg. Frank hxmaturia was present, and the urine contained a large amount of protein. After a week the blood-pressure was normal, and hasmaturia was found on microscopy only. 5 weeks later, when oedema had subsided but some proteinuria persisted, a severe pharyngitis developed and the patient became grossly oedematous with pleural effusions and ascites. The bloodpressure was 120/80 mm. Hg. The results of investigations
blood-urea-nitrogen, 31 mg. per 100 ml.; antistreptolysin titre, 300 Todd units; microscopy of the urinary sediment showed 1-2 leucocytes, 8-12 hyaline and granular casts, and occasional oval fat bodies per high-power field; daily urinary protein excretion, 13-24 g.; serum-albumin, 0-86 g. per 100 ml.; serum-globulin, 2-74 g. per 100 ml.; serum-cholesterol, 940 g. per 100 ml. Renal biopsy subsequently revealed mild focal axial hypercellularity in the glomeruli without apparent significant thickening of peripheral capillary basement membrane. Hicks-Pitney thromboplastin-generation tests gave abnormal results on the day before and 10 days after beginning prednisolone, 50 mg. daily. Factor-Ix assay showed 5°n and 15% activity (see table). Diuresis began on the 17th day of steroid therapy and urinary protein decreased. On the 20th day were:
1080 INITIAL COAGULATION STUDIES
the result of the Hicks-Pitney test was normal although the serum-albumin was still only 1-4 g. per 100 ml. The screeningtest and factor-ix assay results were normal 7 and 8 months
later. Case 3 A boy,
aged 7 years, was admitted with the nephrotic which had developed over 4 days. There was no syndrome sore throat. He had extensive oedema, with ascites preceding and small bilateral pleural effusions. The blood-pressure was 115/70
mm.
Hg.
The results of investigations were: blood-urea-nitrogen, 14 mg. per 100 ml.; antistreptolysin titre, 100 Todd units; throat-culture grew a few &bgr;-haemolytic streptococci and pneumococci. Microscopy of the urinary sediment showed occasional hyaline casts, 1-3 leucocytes per high-power field;
daily urinary protein excretion, 4-2-6 g.; serum-albumin, 0-74 g. per 100 ml.; serum-globulin, 2-66 g. per 100 ml.; serum-cholesterol, 740 mg. per 100 ml. Subsequent renal biopsy showed slight glomerular changes under light microscopy. Coagulation studies revealed a defect in the screening thromboplastin-generation test, and factor-ix assay gave 26% activity. After prednisolone 30 mg. was given daily diuresis began on the 9th day and the daily urinary protein excretion fell to 160 mg. On the 9th day of steroid treatment, assay of factor ix gave 100% activity. Case 4 A boy, aged 14 years, was admitted with insidious painless swelling of the abdomen, face, and legs for the previous 2 weeks without an antecedent sore throat. There was pitting oedema up to the level of the knees, ascites, and bilateral pleural effusions. The blood-pressure was 110/80 mm. Hg. The results of investigations were: blood-urea-nitrogen, 18 mg. per 100 ml.; plasma-creatinine, 1-92 mg. per 100 ml.; creatinine clearance, 39 ml. per minute; antistreptolysin titre, 100 Todd units; throat-culture grew no pathogenic organisms. Microscopy of the urinary sediment showed 5-8 leucocytes, 5-10 erythrocytes, and occasional hyaline and granular casts per high-power
field; daily urinary protein excretion, 6-12 g.; serum-albumin, 0-8 g. per 100 ml.; serumglobulin, 3-3 g. per 100 ml.; serum-cholesterol, 334 mg. per 100 ml. After 8
days of observa-
tion, oral prednisolone 50 mg. daily was begun; proteinuria decreased and diuresis began on the 10th day of therapy (fig. 2). Renal biopsy, soon after diuresis began, revealed no
glomerular basement membrane thickening light microscopy but occasional small proliferative endothelial foci were present. Hicks-Pitney thromboplastin-generation tests under
results, and factor-Ix levels considerably reduced on three occasions before diuresis (see table) but returned to normal promptly thereafter (fig. 2). These gave abnormal were
serial assays were carried out in a separate institution where there was no access to the clinical data.
Results
The results of the original coagulation investigations in each patient are shown in the accompanying table. Subsequent changes in factor-ix assay and in the HicksPitney thromboplastin-generation screening-test in relation to clinical events in cases 1, 2, and 3 are shown in fig. 1, and in case 4 in fig. 2. In cases 3 and 4, tests for circulating anticoagulants which might have inhibited factor ix were negative. Fibrinogen levels were raised in all four patients. Discussion were made in patients with the without advanced clinical or but nephrotic syndrome, biochemical renal failure. Thus, most of the published reports which refer to patients with renal failure and have shown a variety of coagulation defects are not relevant to these cases (Lewis et al. 1956, Cheyney and Bonnin 1962, Castaldi et al. 1966). Yatzidis and Richet (1957), however, described twenty patients with the nephrotic syndrome none of whom had any defect of plasma-coagulation factors, but the urine of sixteen patients contained prothrombin (factor II), and in eleven of these proconvertin (factor vil) was present as well.
These observations
An isolated deficiency of factor ix is uncommon, and usually manifests itself as the hereditary disorder, Christmas disease. None of the patients described had a history that suggested a congenital bleeding diathesis, and coagulation studies on relatives of case 1 were normal. The factor-ix deficiency in each patient discovered at the height of the illness disappeared with remission and has not recurred. Bleeding was absent in all; the observation was based entirely on laboratory coagulation studies.
1081
Patients such as the four described here, with slight changes in the glomerular basement membrane which respond to steroids, tend to lose a large proportion of proteins with relatively low molecular weights. The molecular weight of factor ix has been estimated variously at between 100,000 and 200,000 using a gel-filtration technique (Lewis 1964) and 110,000 using electron irradiation (Aronson et al. 1962). Factor ix is therefore rather larger than the range of protein molecules which cleared most rapidly in the nephrotic syndrome associated with slight glomerular change. It may be significant that the deficiency was found in only four of more than thirty patients with the nephrotic syndrome who were examined, and each patient had a particularly low serum-albumin. An attractive hypothesis is that factor ix was lost in the urine in amounts sufficient to lower plasma levels. The rapid rise to normal of factor-ix levels in the plasma coincident with a decrease in proteinuria is consistent with this hypothesis, but serum-albumin levels revert to normal much more slowly. Our attempts to demonstrate factor ix in urine from several nephrotic patients, including case 4, have so far been inconclusive. Accurate identification of any coagulation factor in the urine is difficult, and nephrotic urine " has definite proteolytic activity " (Yatzidis and Richet 1957). Assay of urine with conventional coagulation techniques may be interfered with by other substances, such as uroplastin, present in the urine (Tocantins and Lindquist 1947, von Kaulla 1956, Joist and Alkjaersig 1966). Because factor ix has not been isolated and purified, it is difficult to develop other assay techniques, but a radioactive immunological method (Adelson et al. 1963) might be applicable. We wish to thank Dr. W. M. Irwin, Dr. J. M. Bonnin, Prof. B. S. Hetzel, and Dr. I. Magarey who referred patients; and Dr. H. Kronenberg who assayed factor ix in case 1 independently. Requests for reprints should be addressed to J. R. L. REFERENCES
Fig. 2-Biochemical changes and factor-IX assay in case 4, indicating response to therapy with prednisolone.
Adelson, E., Rheingold, J. J., Parker, O., Steiner, M., Kirby, J. C. (1963) J. clin. Invest. 42, 1040. Aronson, D. L., Preiss, J. W., Mosesson, M. W. (1962) Thromb. Diath. hœmorrh. 8, 270. Biggs, R., Bidwell, E., Handley, D. A., Macfarlane, R. G., Trueta, J., Elliot-Smith, A., Dike, G. W. R., Ash, B. J. (1961) Br. J. Hœmat. 7, 349. Douglas, A. S. (1953) J. clin. Path. 6, 23. Macfarlane, R. G. (1962) Scientific Publications. Oxford. Blainey, J. D., Brewer, D. B., Hardwicke, J., Soothill, J. F. (1960) Q. Jl Med. 29, 235. Castaldi, P. A., Rozenberg, M. C., Stewart, J. H. (1966) Lancet, ii, 66. Cheyney, K., Bonnin, J. A. (1962) Br. J. Hœmat. 8, 215. Denson, K. W. (1961) Acta hœmat. 25, 105. Gitlin, D., Janeway, C. A., Farr, L. E. (1956) J. clin. Invest. 35, 44. Joist, H., Alkjaersig, N. (1966) XIth Congress International Society of Hæmatology, Sydney. Kark, R. M., Pirani, C. L., Pollak, V. E., Muehrcke, R. C., Blainey, J. D. (1958) Ann. intern. Med. 49, 751. Katz, J., Bonorris, G., Sellers, A. L. (1963) J. Lab. clin. Med. 62, 910. Lewis, J. H. (1964) Proc. Soc. exp. Biol. Med. 116, 120. Zucker, M. B., Ferguson, J. H. (1956) Blood, 11, 1073. MacPherson, J. C., Hardisty, R. M. (1961) Thromb. Diath. hœmorrh. 6, 492. Metcoff, J., Janeway, C. A. (1961) J. Pediat. 58, 640. Pitney, W. R. (1956) Br. J. Hœmat. 2, 250. Tocantins, L. M., Lindquist, J. N. (1947) Proc. Soc. exp. Biol. Med. 65, 44. von Kaulla, K. N. (1956) ibid. 91, 543. Yatzidis, H., Richet, G. (1957) Rev. fr. Étud. clin. biol. 2, 717. —
Repeated assays, however, showed the presence of the defect, and these were confirmed by independent assay carried out in another laboratory; the classical thromboplastin-generation and screening-tests gave abnormal results in three separate laboratories. The possibility that low factor-ix assays were due to inhibition by circulating anticoagulants in the plasma was excluded in cases 3 and 4. The mechanism of the factor-ix deficiency is obscure. The structure and metabolism of factor ix have not been characterised adequately, but it is thought to be a protein produced by the liver. There was no conventional biochemical evidence of liver dysfunction in these patients, and factors 11, v, vil, and x, which originate from the liver, were not deficient. Many aspects of the metabolic disorder in the clinical and experimental nephrotic syndrome remain incompletely explained (Gitlin et al. 1956, Metcoff and Janeway 1961, Katz et al. 1963). In general, the low levels of circulating proteins appear to be due to loss of protein rather than to impaired protein synthesis. Protein is lost in the urine, but significant amounts may also be broken down in the kidney. Blainey et al. (1960) have shown that the character of urinary protein loss can be related to the histological lesion and the clinical response to steroid therapy.
—
—
" ... I am an honest disciple of Karl Popper in believing that the function of hypothesis in science is all-important in providing clear statements that are susceptible of disproof by experimental or observational data, or both. No such thing as absolute truth exists, but there is always contemporary truth in the form of those axioms, generalisations, theories and hypotheses that, in the opinion of competent scholars, have not yet been disproved."-Sir MACFARLANE BURNET, O.M., F.R.S., in Reflections on Research and the Future of Medicine; p. 10. New York and London, 1967.
hypercholesterolxmia is related to excess circulating thyrotrophic hormone, a raised serum-cholesterol would be expected during the time that the failing thyroid was responding adequately to the increasing pituitary stimulus.
Methods
If the
analogy between prediabetes and premyxoedema possible aetiological factors in the development of coronary-artery disease. Thannhauser (1958) repeatedly warns against arguing from the particular to the general when considering the high incidence of atheroma in idiopathic familial hypercholesterolxmia. Nevertheless, factors affecting serum-cholesterol consistently influence the incidence of coronary-artery disease in the same direction. The following factors are associated There may be
an
as
with increased serum-cholesterol and increased incidence of coronary-artery disease: genetic predisposition, sedentary work, obesity, age, male sex, post-menopausal women (compared with premenopausal women), hypertension, idiopathic xanthomatosis, diabetes and prediabetes, and pre-myxoedema. Factors associated with decreased serumcholesterol and incidence of coronary-artery disease: include exercise and manual work, leanness, fasting and starvation, cestrogens, and thyrotoxicosis. There are many aetiological factors in coronary-artery disease. The importance of premyxoedema which might cause death from coronary-artery disease before the myxoedema itself develops can be assessed only by further studies. REFERENCES
Christie, J. F., Lyall, A., Anderson, T. E. (1930) Br. J. Derm. 42, 429. Craig, L. S., Lisser, H. (1944) J. clin. Endocr. Metab. 4, 12. Escamilla, R. (1942) ibid. 2, 33. Fowler, P. B. S., Briggs, J. H. (1962) Proc. R. Soc. Med. 55, 601. Gee, D. J., Goldstein, J., Gray, C. H., Fowler, J. F. (1959) Br. med. J. ii, 341. Sweitzer, S. E., Winer, L. H. (1940) Archs. Derm. Syph. 42, 419. Thannhauser, S. J. (1958) Lipidoses. New York and London. Thorp, J. M. (1962) Lancet, i, 1323. (1963) J. Atheroscler. Res. 3, 351. —
FACTOR-IX DEFICIENCY IN THE NEPHROTIC SYNDROME D. A. HANDLEY M.B. Adelaide, M.R.A.C.P., M.C.Path., M.C.P.A. HÆMATOLOGIST, INSTITUTE OF MEDICAL AND VETERINARY SCIENCE, ADELAIDE
J. R. LAWRENCE M.B.
DIRECTOR,
RENAL
Adelaide, M.R.A.C.P.
UNIT, QUEEN ELIZABETH HOSPITAL, WOODVILLE, SOUTH AUSTRALIA
Four patients with the nephrotic syndrome shown to have a deficiency of circulatfactor IX. In each, the abnormality was ing coagulation reversed when the nephrotic syndrome responded to steroid therapy.
Summary
were
Introduction
MANY
changes in the constituents of circulating blood
have been described in patients with the nephrotic
syndrome (Kark et al. 1958). In this paper we describe four nephrotic patients who were shown to have low levels of circulating coagulation factor ix during the acute phase of the illness which returned to normal with improvement in the nephrotic syndrome. The abnormality was observed for renal
during routine coagulation studies in preparation biopsy. The thromboplastin-generation screening test (Hicks-Pitney) gave abnormal results in each case, and subsequent assay of factor ix identified the deficiency.
The
bleeding-time (Duke), tourniquet test, whole-blood clotting-time (Lee and White), and one-stage " prothrombin "time (Quick) and fibrinogen assay were carried out as described by Biggs and Macfarlane (1962). The thromboplastingeneration screening test of Hicks and Pitney, as modified by MacPherson and Hardisty (1961), was used as a preliminary test of the plasma factors required for the intriasic thromboplastin system. Factor ix was assayed according to the method described by Biggs et al. (1961) which is based on the classical thromboplastin-generation test. Factor x was assayed according ing to the method of Denson (1961), using charcoal-filtered ox plasma as a source of factor-x-deficient substrate. Factor vm was assayed according to the method of Pitney (1956). Circulating anticoagulants affecting factor ix were tested by making mixtures of normal plasma and plasma from the patient. These mixtures were then tested in the Hicks-Pitney screeningtest system and the classical thromboplastin-generation test of Biggs and Douglas (1953). Urinary protein was assayed by the biuret method. Case-records Case 1 A 15-year-old boy was admitted to hospital with swelling of the legs and face. There was no preceding sore throat. He had gross oedema of the legs and face, and ascites. The bloodpressure was 115/65 mm. Hg. The results of investigations were:
titre,
blood-urea-nitrogen, 75 Todd
14 mg. per 100
units; throat culture,
no
ml.; antistreptolysin growth of pathogens.
Microscopic examination of the urinary sediment showed 10-15 hyaline and granular casts per high-power field; daily urinary protein excretion was 7-10 g.; serum-albumin, 0-51 g. per 100 ml.; serum-globulin, 3-1 g. per 100 ml.; serum-cholesterol, 720 mg. per 100 ml. For 2 months the patient was treated with diuretics without improvement in the nephrotic syndrome. He was then assessed for renal biopsy, but a defect was found in the thromboplastin-
generation screening-test. Assay of factor ix showed 25% activity (see table and fig. 1). Prednisolone 40 mg. daily was given and diuresis began on the 11th day. Proteinuria decreased sharply 18 days after steroid therapy was begun, and the serum-albumin rose to 3-0 g. per 100 ml. Subsequently the dose of prednisolone was reduced and then discontinued. There has been no recurrence of proteinuria after 2 years. Further assays of factor ix 6 and 7 months later showed 100% activity. The significance of the nephrotic syndrome in relation to factor-Ix deficiency was not at first appreciated in this patient, although extensive family studies showed no other evidence of Christmas disease. Case 2 A year after case 1 was admitted a farmer, aged 18 years, with acute nephritis which had come on 10 days after a severe sore throat, was admitted. His blood-pressure was 150/90 mm. Hg. Frank hxmaturia was present, and the urine contained a large amount of protein. After a week the blood-pressure was normal, and hasmaturia was found on microscopy only. 5 weeks later, when oedema had subsided but some proteinuria persisted, a severe pharyngitis developed and the patient became grossly oedematous with pleural effusions and ascites. The bloodpressure was 120/80 mm. Hg. The results of investigations
blood-urea-nitrogen, 31 mg. per 100 ml.; antistreptolysin titre, 300 Todd units; microscopy of the urinary sediment showed 1-2 leucocytes, 8-12 hyaline and granular casts, and occasional oval fat bodies per high-power field; daily urinary protein excretion, 13-24 g.; serum-albumin, 0-86 g. per 100 ml.; serum-globulin, 2-74 g. per 100 ml.; serum-cholesterol, 940 g. per 100 ml. Renal biopsy subsequently revealed mild focal axial hypercellularity in the glomeruli without apparent significant thickening of peripheral capillary basement membrane. Hicks-Pitney thromboplastin-generation tests gave abnormal results on the day before and 10 days after beginning prednisolone, 50 mg. daily. Factor-Ix assay showed 5°n and 15% activity (see table). Diuresis began on the 17th day of steroid therapy and urinary protein decreased. On the 20th day were:
1080 INITIAL COAGULATION STUDIES
the result of the Hicks-Pitney test was normal although the serum-albumin was still only 1-4 g. per 100 ml. The screeningtest and factor-ix assay results were normal 7 and 8 months
later. Case 3 A boy,
aged 7 years, was admitted with the nephrotic which had developed over 4 days. There was no syndrome sore throat. He had extensive oedema, with ascites preceding and small bilateral pleural effusions. The blood-pressure was 115/70
mm.
Hg.
The results of investigations were: blood-urea-nitrogen, 14 mg. per 100 ml.; antistreptolysin titre, 100 Todd units; throat-culture grew a few &bgr;-haemolytic streptococci and pneumococci. Microscopy of the urinary sediment showed occasional hyaline casts, 1-3 leucocytes per high-power field;
daily urinary protein excretion, 4-2-6 g.; serum-albumin, 0-74 g. per 100 ml.; serum-globulin, 2-66 g. per 100 ml.; serum-cholesterol, 740 mg. per 100 ml. Subsequent renal biopsy showed slight glomerular changes under light microscopy. Coagulation studies revealed a defect in the screening thromboplastin-generation test, and factor-ix assay gave 26% activity. After prednisolone 30 mg. was given daily diuresis began on the 9th day and the daily urinary protein excretion fell to 160 mg. On the 9th day of steroid treatment, assay of factor ix gave 100% activity. Case 4 A boy, aged 14 years, was admitted with insidious painless swelling of the abdomen, face, and legs for the previous 2 weeks without an antecedent sore throat. There was pitting oedema up to the level of the knees, ascites, and bilateral pleural effusions. The blood-pressure was 110/80 mm. Hg. The results of investigations were: blood-urea-nitrogen, 18 mg. per 100 ml.; plasma-creatinine, 1-92 mg. per 100 ml.; creatinine clearance, 39 ml. per minute; antistreptolysin titre, 100 Todd units; throat-culture grew no pathogenic organisms. Microscopy of the urinary sediment showed 5-8 leucocytes, 5-10 erythrocytes, and occasional hyaline and granular casts per high-power
field; daily urinary protein excretion, 6-12 g.; serum-albumin, 0-8 g. per 100 ml.; serumglobulin, 3-3 g. per 100 ml.; serum-cholesterol, 334 mg. per 100 ml. After 8
days of observa-
tion, oral prednisolone 50 mg. daily was begun; proteinuria decreased and diuresis began on the 10th day of therapy (fig. 2). Renal biopsy, soon after diuresis began, revealed no
glomerular basement membrane thickening light microscopy but occasional small proliferative endothelial foci were present. Hicks-Pitney thromboplastin-generation tests under
results, and factor-Ix levels considerably reduced on three occasions before diuresis (see table) but returned to normal promptly thereafter (fig. 2). These gave abnormal were
serial assays were carried out in a separate institution where there was no access to the clinical data.
Results
The results of the original coagulation investigations in each patient are shown in the accompanying table. Subsequent changes in factor-ix assay and in the HicksPitney thromboplastin-generation screening-test in relation to clinical events in cases 1, 2, and 3 are shown in fig. 1, and in case 4 in fig. 2. In cases 3 and 4, tests for circulating anticoagulants which might have inhibited factor ix were negative. Fibrinogen levels were raised in all four patients. Discussion were made in patients with the without advanced clinical or but nephrotic syndrome, biochemical renal failure. Thus, most of the published reports which refer to patients with renal failure and have shown a variety of coagulation defects are not relevant to these cases (Lewis et al. 1956, Cheyney and Bonnin 1962, Castaldi et al. 1966). Yatzidis and Richet (1957), however, described twenty patients with the nephrotic syndrome none of whom had any defect of plasma-coagulation factors, but the urine of sixteen patients contained prothrombin (factor II), and in eleven of these proconvertin (factor vil) was present as well.
These observations
An isolated deficiency of factor ix is uncommon, and usually manifests itself as the hereditary disorder, Christmas disease. None of the patients described had a history that suggested a congenital bleeding diathesis, and coagulation studies on relatives of case 1 were normal. The factor-ix deficiency in each patient discovered at the height of the illness disappeared with remission and has not recurred. Bleeding was absent in all; the observation was based entirely on laboratory coagulation studies.
1081
Patients such as the four described here, with slight changes in the glomerular basement membrane which respond to steroids, tend to lose a large proportion of proteins with relatively low molecular weights. The molecular weight of factor ix has been estimated variously at between 100,000 and 200,000 using a gel-filtration technique (Lewis 1964) and 110,000 using electron irradiation (Aronson et al. 1962). Factor ix is therefore rather larger than the range of protein molecules which cleared most rapidly in the nephrotic syndrome associated with slight glomerular change. It may be significant that the deficiency was found in only four of more than thirty patients with the nephrotic syndrome who were examined, and each patient had a particularly low serum-albumin. An attractive hypothesis is that factor ix was lost in the urine in amounts sufficient to lower plasma levels. The rapid rise to normal of factor-ix levels in the plasma coincident with a decrease in proteinuria is consistent with this hypothesis, but serum-albumin levels revert to normal much more slowly. Our attempts to demonstrate factor ix in urine from several nephrotic patients, including case 4, have so far been inconclusive. Accurate identification of any coagulation factor in the urine is difficult, and nephrotic urine " has definite proteolytic activity " (Yatzidis and Richet 1957). Assay of urine with conventional coagulation techniques may be interfered with by other substances, such as uroplastin, present in the urine (Tocantins and Lindquist 1947, von Kaulla 1956, Joist and Alkjaersig 1966). Because factor ix has not been isolated and purified, it is difficult to develop other assay techniques, but a radioactive immunological method (Adelson et al. 1963) might be applicable. We wish to thank Dr. W. M. Irwin, Dr. J. M. Bonnin, Prof. B. S. Hetzel, and Dr. I. Magarey who referred patients; and Dr. H. Kronenberg who assayed factor ix in case 1 independently. Requests for reprints should be addressed to J. R. L. REFERENCES
Fig. 2-Biochemical changes and factor-IX assay in case 4, indicating response to therapy with prednisolone.
Adelson, E., Rheingold, J. J., Parker, O., Steiner, M., Kirby, J. C. (1963) J. clin. Invest. 42, 1040. Aronson, D. L., Preiss, J. W., Mosesson, M. W. (1962) Thromb. Diath. hœmorrh. 8, 270. Biggs, R., Bidwell, E., Handley, D. A., Macfarlane, R. G., Trueta, J., Elliot-Smith, A., Dike, G. W. R., Ash, B. J. (1961) Br. J. Hœmat. 7, 349. Douglas, A. S. (1953) J. clin. Path. 6, 23. Macfarlane, R. G. (1962) Scientific Publications. Oxford. Blainey, J. D., Brewer, D. B., Hardwicke, J., Soothill, J. F. (1960) Q. Jl Med. 29, 235. Castaldi, P. A., Rozenberg, M. C., Stewart, J. H. (1966) Lancet, ii, 66. Cheyney, K., Bonnin, J. A. (1962) Br. J. Hœmat. 8, 215. Denson, K. W. (1961) Acta hœmat. 25, 105. Gitlin, D., Janeway, C. A., Farr, L. E. (1956) J. clin. Invest. 35, 44. Joist, H., Alkjaersig, N. (1966) XIth Congress International Society of Hæmatology, Sydney. Kark, R. M., Pirani, C. L., Pollak, V. E., Muehrcke, R. C., Blainey, J. D. (1958) Ann. intern. Med. 49, 751. Katz, J., Bonorris, G., Sellers, A. L. (1963) J. Lab. clin. Med. 62, 910. Lewis, J. H. (1964) Proc. Soc. exp. Biol. Med. 116, 120. Zucker, M. B., Ferguson, J. H. (1956) Blood, 11, 1073. MacPherson, J. C., Hardisty, R. M. (1961) Thromb. Diath. hœmorrh. 6, 492. Metcoff, J., Janeway, C. A. (1961) J. Pediat. 58, 640. Pitney, W. R. (1956) Br. J. Hœmat. 2, 250. Tocantins, L. M., Lindquist, J. N. (1947) Proc. Soc. exp. Biol. Med. 65, 44. von Kaulla, K. N. (1956) ibid. 91, 543. Yatzidis, H., Richet, G. (1957) Rev. fr. Étud. clin. biol. 2, 717. —
Repeated assays, however, showed the presence of the defect, and these were confirmed by independent assay carried out in another laboratory; the classical thromboplastin-generation and screening-tests gave abnormal results in three separate laboratories. The possibility that low factor-ix assays were due to inhibition by circulating anticoagulants in the plasma was excluded in cases 3 and 4. The mechanism of the factor-ix deficiency is obscure. The structure and metabolism of factor ix have not been characterised adequately, but it is thought to be a protein produced by the liver. There was no conventional biochemical evidence of liver dysfunction in these patients, and factors 11, v, vil, and x, which originate from the liver, were not deficient. Many aspects of the metabolic disorder in the clinical and experimental nephrotic syndrome remain incompletely explained (Gitlin et al. 1956, Metcoff and Janeway 1961, Katz et al. 1963). In general, the low levels of circulating proteins appear to be due to loss of protein rather than to impaired protein synthesis. Protein is lost in the urine, but significant amounts may also be broken down in the kidney. Blainey et al. (1960) have shown that the character of urinary protein loss can be related to the histological lesion and the clinical response to steroid therapy.
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" ... I am an honest disciple of Karl Popper in believing that the function of hypothesis in science is all-important in providing clear statements that are susceptible of disproof by experimental or observational data, or both. No such thing as absolute truth exists, but there is always contemporary truth in the form of those axioms, generalisations, theories and hypotheses that, in the opinion of competent scholars, have not yet been disproved."-Sir MACFARLANE BURNET, O.M., F.R.S., in Reflections on Research and the Future of Medicine; p. 10. New York and London, 1967.