1208
PLASMA EXCHANGE IN THE MANAGEMENT OF HOMOZYGOUS FAMILIAL HYPERCHOLESTEROLÆMIA
G. R. THOMPSON
R. LOWENTHAL N. B. MYANT Departments of Medicine and Hœmatology, Royal
Postgraduate Medical School, and Medical Research Lipid Metabolism Unit, Hammersmith Hospital,
Council
London W12 0HS
Two young
with
homozygous hypercholesterolæmia and coronary and aortic atheroma were treated by repeated plasma exchange, using a continuous-flow blood-cell separator, for 4 and 8 months. A pronounced reduction in plasma cholesterol and low-density lipoprotein (L.D.L.) concentrations was achieved by exchanging each patient’s plasma with cholesterol-free plasma protein fraction (B.P.), at 3-weekly intervals on an outpatient basis. By pre-labelling the patients’ cholesterol with 14C and comparing the specific activity of the cholesterol in adipose tissue with that in plasma, evidence was obtained of an influx of tissue cholesterol into plasma after each exchange. There were no side-effects and both patients lost their angina. Plasma exchange offers a new and practical approach to the long-term management of this lethal disorder and may also provide information about the possible reversibility of human Summary familial
women
atheroma.
Introduction FAMILIAL
hypercholesterolaemia (F.H.) (familial typehyperlipoproteinmmia) is characterised by a raised plasma-low-density-lipoprotein (L.D.L.) concentration and is inherited as an incompletely dominant trait. Patients with the homozygous form of the disease die prematurely from myocardial infarction due to coronary atheroma, few surviving beyond the age of 30.1 It has proved extremely difficult to control the grossly elevated plasma-L.D.L. concentration and the accompanying hypercholesterolaemia in these patients. Dietary treatII
alone is of very limited value, but some measure of success has been achieved by the use of cholestyramine,2,3 especially when given in combination with nicotinic acid.4 Ileal bypass has been proposed as an alternative to drug therapy,5 but its superiority remains unproven.6 Recently, Starzl et al.achieved a striking reduction in L.D.L. levels in a young homozygous female patient in whom they had performed a portacaval shunt, but less impressive results were obtained in four other homozygous patients who have since been treated in the same way.s Other forms of therapy which have been partially successful in lowering L.D.L. levels over relatively short periods include the intravenous administration of fat emulsions,9 intravenous alimentation, 7, ’,I and conventional plasmapheresis. 11,12 We describe a new approach to the management of homozygous F.H., which involves repeated plasma exchange by means of a continuous-flow blood-cell separator, performed on an outpatient basis. ment
Patients and Methods Both
patients are young women, weight for their height.
of normal
short in stature, but
Patient 1.-This 21-year-old trainee teacher has been described in detail elsewhere (patient 2 in Moutafis and Myant3 and patient 1 in Moutafis et al.1). She was born of hypercholesterolxmic Greek-Cypriot parents, and cutaneous and tendon xanthomata developed in infancy. Subsequently she was discovered to have hypercholesterolaemia, was referred to one of us (N. B. M.) in 1965, and has since been treated with various drugs, including clofibrate, cholestyramine, nicotinic acid, neomycin, and polidexide Secholex’). Until recently she has had angina of effort, confirmed by a markedly ischaemic-type exercise electrocardiogram (E.C.G.), and she has a bruit and loud systolic murmur over the aortic area. showed a stenotic lesion at the Coronary ostium of the right coronary artery and there was a 35 mm. Hg pressure gradient across the aortic valve. During the past 3 years her serum-cholesterol concentration has ranged from 450 to 1050 mg. per 100 ml., with a mean of approximately 600 mg. per 100 ml.; fasting serumtriglyceride concentrations have always been within the normal range. Lipoprotein electrophoresis and preparative ultracentrifugation show a very striking increase in plasmaL.D.L. concentration. These features are characteristic of with a type-na pattem.13 homozygous F.H., Patient 2 is a 24-year-old housewife, and an only child. Xanthomata in both Achilles tendons were first noticed at about the age of 12, and hypercholesterolsemia was detected 6 years later. Her father died of a myocardial infarct at the age of 32. Her mother, who is of Greek origin, is 51 and, although she has no xanthomata, she has an elevated serum-cholesterol concentration (304 mg. per 100 ml.), with a normal serum-triglyceride concentration. In 1970 the patient was referred to one of us (G. R. T.) and at the time was found to have bilateral xanthomata in the Achilles and extensor-hallucis tendons, together with a bruit and systolic murmur over the aortic area. She had noticed occasional episodes of chest pain on exertion and a recent exercise E.c.G. showed ischxmic changes. Angiography showed partial stenosis of the left coronary ostium and some relative narrowing of the proximal portion of the ascending aorta; there was a 34 mm. Hg pressure gradient across the aortic valve. Her serum-cholesterol concentration ranged between 400 and 800 mg. per 100 ml. and fasting serum-triglycerides between 100 and 250 mg, per 100 ml. Preparative ultraand lipoprotein electrophoresis demonstrated centrifugation a large increase in plasma-L.D.L. concentration, with L.D.L. cholesterol levels of more than 500 mg. per 100 ml., and a slight but definite increase in. plasma-very-low-densitylipoprotein (V.L.D.L.) concentration. These features are compatible with a diagnosis of homozygous F.H., with a type ub pattem.13 Treatment with clofibrate, together with either neomycin, cholestyramine, or polidexide, did not control her hypercholesterolsemia adequately.
angiography
Plasma Exchange This was carried out with a continuous-flow blood-cell separator (’ Celltrifuge’, American Instrument Company), using a technique similar to that described by Lowenthal et all but modified for the collection of plasma rather than leucocytes. Dextran was not added to the input line. Plasma exchange was performed on an outpatient basis for 3-4 hours, at approximately 3-weekly intervals. On each occasion 2-4-5 litres of each patient’s plasma was replaced with an equivalent volume of either groupspecific fresh-frozen plasma (F.F.P.) or plasma protein fraction (B.P.) (P.P.F.) When the latter was used, 1-5 meq. of potassium chloride and 2 g. of calcium gluconate were added to each 400 ml. bottle before infusion, to compensate for the low potassium and calcium content of P.P.F. 6-12 weeks before undergoing her first plasma exchange, each patient received 50 1ACi of [14C] -cholesterol intra-
1209
yenously,
as previously described.3 The specific activity of [1C]-cholesterol was assayed in fat-biopsy specimens taken from the vicinity of the iliac crest on the day preceding the first plasma exchange, and was also assayed in subsequent samples of plasma.
Analytical
Methods
Cholesterol was assayed by the method of Abell et al.,ls 14C-radioactivity being assayed by counting an aliquot of the hexane extract of the saponified sample. Triglyceride L.D.L. and was assayed by an autoanalyser technique.16 were measured cholesterol high-density lipoprotein (H.D.L.) and the in supernatant, respectively, obtained precipitate after the addition of 1 volume of 1 % dextran sulphate in 1M calcium chloride to 10 volumes of the fraction of plasma with a density greater than 1’006.17
Results
Plasma-cholesterol and Changes in Specific Activity Patient 1 has had eleven plasma exchanges during the past 8 months. This has reduced her plasmacholesterol concentration from its pre-exchange level of 795 mg. per 100 ml. to an average value of 370 mg. per 100 ml. (fig. 1). P.P.F. consists mainly of albumin and contains no cholesterol, and thus produces a’ greater immediate reduction in plasma-cholesterol concentration than F.F.P., which contains on average 230 mg. per 100 ml. cholesterol. Almost 70 g. of cholesterol has been removed to date, mainly as L.D.L. (fig. 1). Plasma-cholesterol rose more rapidly during the period when drug therapy was discontinued. To determine whether plasma exchange resulted in mobilisation of tissue cholesterol, changes in the specific activity of plasma-cholesterol were measured (fig. 2). Initially the specific activity of [l4C] -cholesterol was 10 times higher in adipose tissue than in plasma. Two consecutive exchanges with F.F.P. resulted in a sharp fall in plasma-specific-activity, followed a few days later by a distinct rise. There was a similar rebound after the third exchange, and on
1974 Fig. 2-Effect of plasma exchange on plasma cholesterol and E14 C]-cholesterol specific activity in patient 1. Asterisk shows specific activity of adipose-tissue cholesterol.
this occasion the rise in specific activity coincided with an increase in plasma-cholesterol, indicating a net influx of high-specific-activity cholesterol into
plasma: Patient 2 has had four plasma exchanges with P.P.F., this being supplemented with F.F.P. on the first occasion only. These resulted in the removal of 30 g. of lipoprotein cholesterol over 4 months. Changes in plasma-cholesterol are shown
_____
fig. 3, the rate of increase again being more rapid during the period when drug therapy was discontinued. In contrast to patient 1, the initial specific activity of adipose-tissue cholesterol was less than twice that of plasma, and the subsequent fall in plasma-specific-activity was much less, presumably because P.P.F. is virtually cholesterol-free. However, again there was a similar, although slighter, rebound in plasma-specific-activity after each exchange, coincident with an increasing plasma-cholesterol concentration. This is further evidence of a net input of cholesterol from tissues into plasma after plasma exchange. In contrast, the rapidly progressive decrease in specific activity which started a week after the first exchange of patient 2 probably represented a temporary increase in cholesterol synthesis. in
Fig. l—ESect of plasma exchange in patient 1.
with F.F.P.
or
P.P.F.
Cumulative total of cholesterol removed is also shown.
on
plasma-cholesterol
Lipoprotein Changes The effect of a single
2-litre
exchange
1210
and calcium supplements to P.P.F. There was no significant decrease in serum calcium or potassium or in hsemoglobin level or leucocyte-count, after a 2 litre P.P.F. exchange. However, the platelet-count fell by up to 50% and there was a twofold prolongation of the prothrombin-time, due to a generalised decrease in clottingfactors. There has been no evidence of spontaneous or prolonged bleeding, but because patients are heparinised during the procedure they are observed for an hour afterwards.
routine addition of
potassium
Discussion The cause of the raised levels of L.D.L. in F.H. is the subject of intense interest. Turnover studies in which the protein
moiety (apoL.D.L.) of i
Asterisk shows
on
specific activity
plasma cholesterol and [lC]-cholesterol
of
adipose-tissue cholesterol.
with P.P.F. on the plasma concentrations of L.D.L. and H.D.L. cholesterol in both patients is shown in fig. 4. Initially there was a striking decrease in the levels of both, but the subsequent gradual and continuing rise in L.D.L. contrasts with the rapid return to preexchange levels and sustained plateau of H.D.L.
Other
Effects ofPlasma Exchange The procedure, which lasts about 3 hours, was well tolerated by both patients. During the earlier exchanges there were some transient episodes of hypotension and hypocalcsemia, but these ceased with the
°
Fig. 4-Levels of L.D.L. and H.D.L. cholesterol in both patients after a single plasma exchange with 2 litres of P.P.F.
labelled
,
suggest that defective catabolism 1$of apoL.D.L. is primarily responsible, although excess
Fig. 3-Effect of plasma exchange specific activity in patient 2.
L.D.L. was
synthesis
may
play a contributory
role 19 In-vitro studies by Goldstein and Brown 20 strongly support the existence of a defect of apoL.D.L. catabolism which, as
these workers have shown, may be associated with the almost total absence of specific L.D.L. receptor sites on the surfaces of cultured skin fibroblasts from homozygous F.H. patients. A further consequence of this defective binding is that F.H. cells do not show the suppression of cholesterol synthesis which occurs when normal cells are incubated with L.D.L. 21,22 The main aim of the present study was to devise a means of reducing the markedly raised plasma-L.D.L. levels which persisted in both patients despite intensive drug therapy. Our results show that by , repeatedly exchanging the plasma of these homozygous F.H. patients with P.P.F. their L.D.L. levels were reduced to values which fall within the range usually found in F.H. heterozygotes. The feasibility of using plasma exchange to lower the serum-cholesterol concentration on a long-term basis and its superiority over conventional plasmapheresis was first demonstrated by Turnberg et al 23 in a patient with xanthomatous neuropathy secondary to biliary cirrhosis. The ease with which this procedure can be carried out is largely due to the ability of continuous-flow blood-cell separators to separate plasma from red and white blood-cells at flow-rates of 30-120 ml. per minute. The rate at which L.D.L. re-accumulates seems to be slower if plasma exchange is used in conjunction with oral hypocholesterolEemic agents. So far there have been no serious side-effects in either of our patients, although a careful watch is being kept on their prothrombintimes. On the contrary, both patients feel remarkablyf well and claim to have lost their angina. A secondary aim of the present study was to assess whether long-term reduction of L.D.L. levels in these two patients will result in regression of their existing atheroma. We intend to repeat the coronary angiograms and also to measure again the pressure gradient across their aortic valves. Starzl et al.2 demonstrated that the decrease in plasma-L.D.L. levels
1211
which followed the creation of a portacaval shunt in their patient was associated with pronounced regression of her atheroma. In an attempt to obtain some this question we pre-labelled patients with 14C so as to obtain a higher specific activity in the tissues than in the plasma at the time of the initial plasma exchanges, using subcutaneous fat as an index of the specific activity of tissue cholesterol. Plasma-specific-activity fell immediately after plasma exchange and then rose again. To some extent this may simply have been a reflection of exchange between labelled cholesterol in tissues and unlabelled cholesterol in plasma. However, the fact that the rise in specific activity coincided with an increase in plasma-cholesterol suggests that there was a net transfer of cholesterol from tissues into plasma, as has been previously observed when the plasmacholesterol has been acutely lowered by other means.26,27 Although the specific-activity data suggest a transfer of cholesterol into plasma, they give no
preliminary
data
cholesterol in
on
our
indication of the tissue from which this was derived. However, there is a slow turnover of cholesterol in atheromatous plaques,2g and plasma exchange may possibly stimulate this process. In view of suggestions that H.D.L. is involved in the transport of cholesterol from tissues into plasma,29 the rapidity with which H.D.L. levels return to normal after plasma exchange is of interest. We thank Dr D. A. G. Galton, Dr J. Goldman, Dr N. Buskard, and Miss Ann Stevenson for help with the cell separator, and Mr A. V. Jadhav for technical assistance; and Prof. R. Steiner, Dr M. Raphael, and Dr C. Oakley for the cardiac catheter and angiographic data. Requests for reprints should be addressed to G. R. T., Medical Research Council Lipid Metabolism Unit, Hammersmith Hospital, Ducane Road, London W12 OHS. REFERENCES 1. Fredrickson, D. S., Levy, R. I. in The Metabolic Basis of Inherited Disease (edited by J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson); p. 545. New York, 1972. 2. Khachadurian, A. K. J. Atheroscler. Res. 1968, 8, 177. 3. Moutafis, C. D., Myant, N. B. Clin. Sci. 1969, 37, 443. 4. Moutafis, C. D., Myant, N. B., Mancini, M., Oriente, P. Atherosclerosis, 1971, 14, 247. 5. Buchwald, H., Moore, R. B., Varco, R. L. Ann. Surg. 1974, 180, 384. 6. Thompson, G. R., Gotto, A. M. Lancet, 1973, ii, 35. 7. Starzl, T. E., Chase, H. P., Putnam, C. W., Porter, K. A. ibid. p. 940. 8. Stein, E. A., Pettifor, J., Mieny, C., Heimann, K. W., Spitz, L., Bersohn, I., Saaron, I., Dinner, M. ibid. 1975, i, 832. 9. Lever, W. F., Waddell, W. R. J. invest. Derm. 1955, 25, 233. 10. Torsvik, H., Feldman, H. A., Fisher, J. E., Lees, R. S. Lancet, 1975, i, 601. 11. De Gennes, J.-L., Touraine, R., Maunand, B., Truffert, J., Laudat, P. Bull. mem. Soc. Hôp Paris, 1967, 118, 1377. 12. Apstein, C. S., George, P. K., Zilversmit, D. B., Feldman, H. A., Lees, R. S. Clin. Res. 1974, 22, 459A. 13. Beaumont, J. L., Carlson, L. A., Cooper, G. R., Fejfar, Z., Fredrickson, D. S., Strasser, T. Wld Hlth Org. Bull. 1970, 43, 891. 14. Lowenthal, R. M., Grossman, L., Goldman, J. M., Storring, R. A., Buskard, N. A., Park, D. S., Murphy, B. C., Spiers, A. S. D., Galton, D. A. G. Lancet, 1975, i, 353. 15. Abell, L. L., Levy, B. B., Brodie, B. B., Kendal, F. E. J. biol. Chem. 1952, 195, 357. 16. Lewis, B., Chait, A., Wootton, I. D. P., Oakley, C. M., Krikler, D. M., Sigurdsson, G., February, A., Maurer, B., Birkhead, J. Lancet, 1974, i, 141. 17. Burstein, M., Scholnick, H. R. Adv. Lipid Res. 1973, 11, 67. 18. Langer, T., Strober, W., Levy, R. I. J. clin. Invest. 1972, 51, 1528. 19. Reichl, D., Simons, L. A., Myant, N. B. Clin. Sci. molec. Med. 1974, 47, 635. 20. Goldstein, J. L., Brown, M. S. J. biol. Chem. 1974, 249, 5153. 21. Goldstein, J. L., Brown, M. S. Proc. natn. Acad. Sci. U.S.A. 1973,
70,
2804.
RELATIVE EFFICACY OF BLOOD, URINE, RECTAL SWAB, BONE-MARROW, AND ROSE-SPOT CULTURES FOR RECOVERY OF SALMONELLA TYPHI IN TYPHOID FEVER ROBERT H. GILMAN MIGUEL TERMINEL MYRON M. LEVINE PABLO HERNANDEZ-MENDOZA RICHARD B. HORNICK Division School
of Infectious Diseases, University of Maryland of Medicine, Baltimore, Maryland, U.S.A., and Hospital Infectiologico C.M. La Raza Hospital, Mexico City, Mexico
The recovery of Salmonella typhi from blood, rectal swab, urine, bone-marrow, and rose spots was compared in 62 patients with typhoid fever, most of whom had received some antibiotic therapy before presentation. S. typhi was isolated from culture of bone-marrow in 56 patients (90%); in contrast, S. typhi was recovered from blood in only 25 (40%), from stool in 23 (37%), and urine in 4 (7%). S. typhi was isolated from 24 (63%) of 38 patients who had rose-spot cultures. If culture sites had been limited to blood, stool, and urine, the bacteriological diagnosis would have been missed in 24 patients. Introduction
Summary
CONFIRMATION of acute typhoid fever requires bacteriological isolation of Salmonella typhi.1-1 In the untreated patient, culture of blood is especially helpful and is positive in about 80% of cases.3—5 However, febrile patients who live or have travelled in endemic areas often present with a history of having already received some form of antibiotic therapy. Prior administration of antibiotics could hinder the bacteriological diagnosis of typhoid fever in a febrile
patient. During
a controlled trial of amoxicillin and cotrimoxazole (trimethoprim and sulphamethoxazole) in the treatment of typhoid fever in Mexico,6 we took the opportunity to compare prospectively the relative efficacy of cultures from various sites for recovery of S. typhi in a population in whom prior self-therapy with antibiotics was common.
Patients and Methods Patients admitted to hospital with a clinical and bacterioof acute typhoid fever and capable of
logical diagnosis
22. Brown, M. S., Goldstein, J. L. ibid. 1974, 71, 788. 23. Turnberg, L. A., Mahoney, M. P., Gleeson, M. H., Freeman, C. B., Gowenlock, A. H. Gut, 1972, 13, 976. 24. Starzl, T. E., Chase, H. P., Putnam, C. W., Nora, J. J. Lancet, 1974, ii, 714. 25. Starzl, T. E., Chase, H. P., Putnam, C. W., Nora, J. J., Fennell, R. H., Porter, K. A. ibid. p. 1263. 26. Grundy, S. M., Ahrens, E. H., Jr., Salen, G., Schreibman, P. H., Nestel, P. J. J. Lipid Res. 1972, 13, 531. 27. Sodhi, H. S., Kudchodkar, B. J., Horlick, L. Atherosclerosis, 1973,
17, 1. 28. 29.
Jagannathan, S. N., Connor, W. E., Baker, W. H., Bhattacharyya, A. K. J. clin. Invest. 1974, 54, 366. Miller, G. J., Miller, N. E. Lancet, 1975, i, 16.
PLASMA EXCHANGE IN THE MANAGEMENT OF HOMOZYGOUS FAMILIAL HYPERCHOLESTEROLÆMIA
G. R. THOMPSON
R. LOWENTHAL N. B. MYANT Departments of Medicine and Hœmatology, Royal
Postgraduate Medical School, and Medical Research Lipid Metabolism Unit, Hammersmith Hospital,
Council
London W12 0HS
Two young
with
homozygous hypercholesterolæmia and coronary and aortic atheroma were treated by repeated plasma exchange, using a continuous-flow blood-cell separator, for 4 and 8 months. A pronounced reduction in plasma cholesterol and low-density lipoprotein (L.D.L.) concentrations was achieved by exchanging each patient’s plasma with cholesterol-free plasma protein fraction (B.P.), at 3-weekly intervals on an outpatient basis. By pre-labelling the patients’ cholesterol with 14C and comparing the specific activity of the cholesterol in adipose tissue with that in plasma, evidence was obtained of an influx of tissue cholesterol into plasma after each exchange. There were no side-effects and both patients lost their angina. Plasma exchange offers a new and practical approach to the long-term management of this lethal disorder and may also provide information about the possible reversibility of human Summary familial
women
atheroma.
Introduction FAMILIAL
hypercholesterolaemia (F.H.) (familial typehyperlipoproteinmmia) is characterised by a raised plasma-low-density-lipoprotein (L.D.L.) concentration and is inherited as an incompletely dominant trait. Patients with the homozygous form of the disease die prematurely from myocardial infarction due to coronary atheroma, few surviving beyond the age of 30.1 It has proved extremely difficult to control the grossly elevated plasma-L.D.L. concentration and the accompanying hypercholesterolaemia in these patients. Dietary treatII
alone is of very limited value, but some measure of success has been achieved by the use of cholestyramine,2,3 especially when given in combination with nicotinic acid.4 Ileal bypass has been proposed as an alternative to drug therapy,5 but its superiority remains unproven.6 Recently, Starzl et al.achieved a striking reduction in L.D.L. levels in a young homozygous female patient in whom they had performed a portacaval shunt, but less impressive results were obtained in four other homozygous patients who have since been treated in the same way.s Other forms of therapy which have been partially successful in lowering L.D.L. levels over relatively short periods include the intravenous administration of fat emulsions,9 intravenous alimentation, 7, ’,I and conventional plasmapheresis. 11,12 We describe a new approach to the management of homozygous F.H., which involves repeated plasma exchange by means of a continuous-flow blood-cell separator, performed on an outpatient basis. ment
Patients and Methods Both
patients are young women, weight for their height.
of normal
short in stature, but
Patient 1.-This 21-year-old trainee teacher has been described in detail elsewhere (patient 2 in Moutafis and Myant3 and patient 1 in Moutafis et al.1). She was born of hypercholesterolxmic Greek-Cypriot parents, and cutaneous and tendon xanthomata developed in infancy. Subsequently she was discovered to have hypercholesterolaemia, was referred to one of us (N. B. M.) in 1965, and has since been treated with various drugs, including clofibrate, cholestyramine, nicotinic acid, neomycin, and polidexide Secholex’). Until recently she has had angina of effort, confirmed by a markedly ischaemic-type exercise electrocardiogram (E.C.G.), and she has a bruit and loud systolic murmur over the aortic area. showed a stenotic lesion at the Coronary ostium of the right coronary artery and there was a 35 mm. Hg pressure gradient across the aortic valve. During the past 3 years her serum-cholesterol concentration has ranged from 450 to 1050 mg. per 100 ml., with a mean of approximately 600 mg. per 100 ml.; fasting serumtriglyceride concentrations have always been within the normal range. Lipoprotein electrophoresis and preparative ultracentrifugation show a very striking increase in plasmaL.D.L. concentration. These features are characteristic of with a type-na pattem.13 homozygous F.H., Patient 2 is a 24-year-old housewife, and an only child. Xanthomata in both Achilles tendons were first noticed at about the age of 12, and hypercholesterolsemia was detected 6 years later. Her father died of a myocardial infarct at the age of 32. Her mother, who is of Greek origin, is 51 and, although she has no xanthomata, she has an elevated serum-cholesterol concentration (304 mg. per 100 ml.), with a normal serum-triglyceride concentration. In 1970 the patient was referred to one of us (G. R. T.) and at the time was found to have bilateral xanthomata in the Achilles and extensor-hallucis tendons, together with a bruit and systolic murmur over the aortic area. She had noticed occasional episodes of chest pain on exertion and a recent exercise E.c.G. showed ischxmic changes. Angiography showed partial stenosis of the left coronary ostium and some relative narrowing of the proximal portion of the ascending aorta; there was a 34 mm. Hg pressure gradient across the aortic valve. Her serum-cholesterol concentration ranged between 400 and 800 mg. per 100 ml. and fasting serum-triglycerides between 100 and 250 mg, per 100 ml. Preparative ultraand lipoprotein electrophoresis demonstrated centrifugation a large increase in plasma-L.D.L. concentration, with L.D.L. cholesterol levels of more than 500 mg. per 100 ml., and a slight but definite increase in. plasma-very-low-densitylipoprotein (V.L.D.L.) concentration. These features are compatible with a diagnosis of homozygous F.H., with a type ub pattem.13 Treatment with clofibrate, together with either neomycin, cholestyramine, or polidexide, did not control her hypercholesterolsemia adequately.
angiography
Plasma Exchange This was carried out with a continuous-flow blood-cell separator (’ Celltrifuge’, American Instrument Company), using a technique similar to that described by Lowenthal et all but modified for the collection of plasma rather than leucocytes. Dextran was not added to the input line. Plasma exchange was performed on an outpatient basis for 3-4 hours, at approximately 3-weekly intervals. On each occasion 2-4-5 litres of each patient’s plasma was replaced with an equivalent volume of either groupspecific fresh-frozen plasma (F.F.P.) or plasma protein fraction (B.P.) (P.P.F.) When the latter was used, 1-5 meq. of potassium chloride and 2 g. of calcium gluconate were added to each 400 ml. bottle before infusion, to compensate for the low potassium and calcium content of P.P.F. 6-12 weeks before undergoing her first plasma exchange, each patient received 50 1ACi of [14C] -cholesterol intra-
1209
yenously,
as previously described.3 The specific activity of [1C]-cholesterol was assayed in fat-biopsy specimens taken from the vicinity of the iliac crest on the day preceding the first plasma exchange, and was also assayed in subsequent samples of plasma.
Analytical
Methods
Cholesterol was assayed by the method of Abell et al.,ls 14C-radioactivity being assayed by counting an aliquot of the hexane extract of the saponified sample. Triglyceride L.D.L. and was assayed by an autoanalyser technique.16 were measured cholesterol high-density lipoprotein (H.D.L.) and the in supernatant, respectively, obtained precipitate after the addition of 1 volume of 1 % dextran sulphate in 1M calcium chloride to 10 volumes of the fraction of plasma with a density greater than 1’006.17
Results
Plasma-cholesterol and Changes in Specific Activity Patient 1 has had eleven plasma exchanges during the past 8 months. This has reduced her plasmacholesterol concentration from its pre-exchange level of 795 mg. per 100 ml. to an average value of 370 mg. per 100 ml. (fig. 1). P.P.F. consists mainly of albumin and contains no cholesterol, and thus produces a’ greater immediate reduction in plasma-cholesterol concentration than F.F.P., which contains on average 230 mg. per 100 ml. cholesterol. Almost 70 g. of cholesterol has been removed to date, mainly as L.D.L. (fig. 1). Plasma-cholesterol rose more rapidly during the period when drug therapy was discontinued. To determine whether plasma exchange resulted in mobilisation of tissue cholesterol, changes in the specific activity of plasma-cholesterol were measured (fig. 2). Initially the specific activity of [l4C] -cholesterol was 10 times higher in adipose tissue than in plasma. Two consecutive exchanges with F.F.P. resulted in a sharp fall in plasma-specific-activity, followed a few days later by a distinct rise. There was a similar rebound after the third exchange, and on
1974 Fig. 2-Effect of plasma exchange on plasma cholesterol and E14 C]-cholesterol specific activity in patient 1. Asterisk shows specific activity of adipose-tissue cholesterol.
this occasion the rise in specific activity coincided with an increase in plasma-cholesterol, indicating a net influx of high-specific-activity cholesterol into
plasma: Patient 2 has had four plasma exchanges with P.P.F., this being supplemented with F.F.P. on the first occasion only. These resulted in the removal of 30 g. of lipoprotein cholesterol over 4 months. Changes in plasma-cholesterol are shown
_____
fig. 3, the rate of increase again being more rapid during the period when drug therapy was discontinued. In contrast to patient 1, the initial specific activity of adipose-tissue cholesterol was less than twice that of plasma, and the subsequent fall in plasma-specific-activity was much less, presumably because P.P.F. is virtually cholesterol-free. However, again there was a similar, although slighter, rebound in plasma-specific-activity after each exchange, coincident with an increasing plasma-cholesterol concentration. This is further evidence of a net input of cholesterol from tissues into plasma after plasma exchange. In contrast, the rapidly progressive decrease in specific activity which started a week after the first exchange of patient 2 probably represented a temporary increase in cholesterol synthesis. in
Fig. l—ESect of plasma exchange in patient 1.
with F.F.P.
or
P.P.F.
Cumulative total of cholesterol removed is also shown.
on
plasma-cholesterol
Lipoprotein Changes The effect of a single
2-litre
exchange
1210
and calcium supplements to P.P.F. There was no significant decrease in serum calcium or potassium or in hsemoglobin level or leucocyte-count, after a 2 litre P.P.F. exchange. However, the platelet-count fell by up to 50% and there was a twofold prolongation of the prothrombin-time, due to a generalised decrease in clottingfactors. There has been no evidence of spontaneous or prolonged bleeding, but because patients are heparinised during the procedure they are observed for an hour afterwards.
routine addition of
potassium
Discussion The cause of the raised levels of L.D.L. in F.H. is the subject of intense interest. Turnover studies in which the protein
moiety (apoL.D.L.) of i
Asterisk shows
on
specific activity
plasma cholesterol and [lC]-cholesterol
of
adipose-tissue cholesterol.
with P.P.F. on the plasma concentrations of L.D.L. and H.D.L. cholesterol in both patients is shown in fig. 4. Initially there was a striking decrease in the levels of both, but the subsequent gradual and continuing rise in L.D.L. contrasts with the rapid return to preexchange levels and sustained plateau of H.D.L.
Other
Effects ofPlasma Exchange The procedure, which lasts about 3 hours, was well tolerated by both patients. During the earlier exchanges there were some transient episodes of hypotension and hypocalcsemia, but these ceased with the
°
Fig. 4-Levels of L.D.L. and H.D.L. cholesterol in both patients after a single plasma exchange with 2 litres of P.P.F.
labelled
,
suggest that defective catabolism 1$of apoL.D.L. is primarily responsible, although excess
Fig. 3-Effect of plasma exchange specific activity in patient 2.
L.D.L. was
synthesis
may
play a contributory
role 19 In-vitro studies by Goldstein and Brown 20 strongly support the existence of a defect of apoL.D.L. catabolism which, as
these workers have shown, may be associated with the almost total absence of specific L.D.L. receptor sites on the surfaces of cultured skin fibroblasts from homozygous F.H. patients. A further consequence of this defective binding is that F.H. cells do not show the suppression of cholesterol synthesis which occurs when normal cells are incubated with L.D.L. 21,22 The main aim of the present study was to devise a means of reducing the markedly raised plasma-L.D.L. levels which persisted in both patients despite intensive drug therapy. Our results show that by , repeatedly exchanging the plasma of these homozygous F.H. patients with P.P.F. their L.D.L. levels were reduced to values which fall within the range usually found in F.H. heterozygotes. The feasibility of using plasma exchange to lower the serum-cholesterol concentration on a long-term basis and its superiority over conventional plasmapheresis was first demonstrated by Turnberg et al 23 in a patient with xanthomatous neuropathy secondary to biliary cirrhosis. The ease with which this procedure can be carried out is largely due to the ability of continuous-flow blood-cell separators to separate plasma from red and white blood-cells at flow-rates of 30-120 ml. per minute. The rate at which L.D.L. re-accumulates seems to be slower if plasma exchange is used in conjunction with oral hypocholesterolEemic agents. So far there have been no serious side-effects in either of our patients, although a careful watch is being kept on their prothrombintimes. On the contrary, both patients feel remarkablyf well and claim to have lost their angina. A secondary aim of the present study was to assess whether long-term reduction of L.D.L. levels in these two patients will result in regression of their existing atheroma. We intend to repeat the coronary angiograms and also to measure again the pressure gradient across their aortic valves. Starzl et al.2 demonstrated that the decrease in plasma-L.D.L. levels
1211
which followed the creation of a portacaval shunt in their patient was associated with pronounced regression of her atheroma. In an attempt to obtain some this question we pre-labelled patients with 14C so as to obtain a higher specific activity in the tissues than in the plasma at the time of the initial plasma exchanges, using subcutaneous fat as an index of the specific activity of tissue cholesterol. Plasma-specific-activity fell immediately after plasma exchange and then rose again. To some extent this may simply have been a reflection of exchange between labelled cholesterol in tissues and unlabelled cholesterol in plasma. However, the fact that the rise in specific activity coincided with an increase in plasma-cholesterol suggests that there was a net transfer of cholesterol from tissues into plasma, as has been previously observed when the plasmacholesterol has been acutely lowered by other means.26,27 Although the specific-activity data suggest a transfer of cholesterol into plasma, they give no
preliminary
data
cholesterol in
on
our
indication of the tissue from which this was derived. However, there is a slow turnover of cholesterol in atheromatous plaques,2g and plasma exchange may possibly stimulate this process. In view of suggestions that H.D.L. is involved in the transport of cholesterol from tissues into plasma,29 the rapidity with which H.D.L. levels return to normal after plasma exchange is of interest. We thank Dr D. A. G. Galton, Dr J. Goldman, Dr N. Buskard, and Miss Ann Stevenson for help with the cell separator, and Mr A. V. Jadhav for technical assistance; and Prof. R. Steiner, Dr M. Raphael, and Dr C. Oakley for the cardiac catheter and angiographic data. Requests for reprints should be addressed to G. R. T., Medical Research Council Lipid Metabolism Unit, Hammersmith Hospital, Ducane Road, London W12 OHS. REFERENCES 1. Fredrickson, D. S., Levy, R. I. in The Metabolic Basis of Inherited Disease (edited by J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson); p. 545. New York, 1972. 2. Khachadurian, A. K. J. Atheroscler. Res. 1968, 8, 177. 3. Moutafis, C. D., Myant, N. B. Clin. Sci. 1969, 37, 443. 4. Moutafis, C. D., Myant, N. B., Mancini, M., Oriente, P. Atherosclerosis, 1971, 14, 247. 5. Buchwald, H., Moore, R. B., Varco, R. L. Ann. Surg. 1974, 180, 384. 6. Thompson, G. R., Gotto, A. M. Lancet, 1973, ii, 35. 7. Starzl, T. E., Chase, H. P., Putnam, C. W., Porter, K. A. ibid. p. 940. 8. Stein, E. A., Pettifor, J., Mieny, C., Heimann, K. W., Spitz, L., Bersohn, I., Saaron, I., Dinner, M. ibid. 1975, i, 832. 9. Lever, W. F., Waddell, W. R. J. invest. Derm. 1955, 25, 233. 10. Torsvik, H., Feldman, H. A., Fisher, J. E., Lees, R. S. Lancet, 1975, i, 601. 11. De Gennes, J.-L., Touraine, R., Maunand, B., Truffert, J., Laudat, P. Bull. mem. Soc. Hôp Paris, 1967, 118, 1377. 12. Apstein, C. S., George, P. K., Zilversmit, D. B., Feldman, H. A., Lees, R. S. Clin. Res. 1974, 22, 459A. 13. Beaumont, J. L., Carlson, L. A., Cooper, G. R., Fejfar, Z., Fredrickson, D. S., Strasser, T. Wld Hlth Org. Bull. 1970, 43, 891. 14. Lowenthal, R. M., Grossman, L., Goldman, J. M., Storring, R. A., Buskard, N. A., Park, D. S., Murphy, B. C., Spiers, A. S. D., Galton, D. A. G. Lancet, 1975, i, 353. 15. Abell, L. L., Levy, B. B., Brodie, B. B., Kendal, F. E. J. biol. Chem. 1952, 195, 357. 16. Lewis, B., Chait, A., Wootton, I. D. P., Oakley, C. M., Krikler, D. M., Sigurdsson, G., February, A., Maurer, B., Birkhead, J. Lancet, 1974, i, 141. 17. Burstein, M., Scholnick, H. R. Adv. Lipid Res. 1973, 11, 67. 18. Langer, T., Strober, W., Levy, R. I. J. clin. Invest. 1972, 51, 1528. 19. Reichl, D., Simons, L. A., Myant, N. B. Clin. Sci. molec. Med. 1974, 47, 635. 20. Goldstein, J. L., Brown, M. S. J. biol. Chem. 1974, 249, 5153. 21. Goldstein, J. L., Brown, M. S. Proc. natn. Acad. Sci. U.S.A. 1973,
70,
2804.
RELATIVE EFFICACY OF BLOOD, URINE, RECTAL SWAB, BONE-MARROW, AND ROSE-SPOT CULTURES FOR RECOVERY OF SALMONELLA TYPHI IN TYPHOID FEVER ROBERT H. GILMAN MIGUEL TERMINEL MYRON M. LEVINE PABLO HERNANDEZ-MENDOZA RICHARD B. HORNICK Division School
of Infectious Diseases, University of Maryland of Medicine, Baltimore, Maryland, U.S.A., and Hospital Infectiologico C.M. La Raza Hospital, Mexico City, Mexico
The recovery of Salmonella typhi from blood, rectal swab, urine, bone-marrow, and rose spots was compared in 62 patients with typhoid fever, most of whom had received some antibiotic therapy before presentation. S. typhi was isolated from culture of bone-marrow in 56 patients (90%); in contrast, S. typhi was recovered from blood in only 25 (40%), from stool in 23 (37%), and urine in 4 (7%). S. typhi was isolated from 24 (63%) of 38 patients who had rose-spot cultures. If culture sites had been limited to blood, stool, and urine, the bacteriological diagnosis would have been missed in 24 patients. Introduction
Summary
CONFIRMATION of acute typhoid fever requires bacteriological isolation of Salmonella typhi.1-1 In the untreated patient, culture of blood is especially helpful and is positive in about 80% of cases.3—5 However, febrile patients who live or have travelled in endemic areas often present with a history of having already received some form of antibiotic therapy. Prior administration of antibiotics could hinder the bacteriological diagnosis of typhoid fever in a febrile
patient. During
a controlled trial of amoxicillin and cotrimoxazole (trimethoprim and sulphamethoxazole) in the treatment of typhoid fever in Mexico,6 we took the opportunity to compare prospectively the relative efficacy of cultures from various sites for recovery of S. typhi in a population in whom prior self-therapy with antibiotics was common.
Patients and Methods Patients admitted to hospital with a clinical and bacterioof acute typhoid fever and capable of
logical diagnosis
22. Brown, M. S., Goldstein, J. L. ibid. 1974, 71, 788. 23. Turnberg, L. A., Mahoney, M. P., Gleeson, M. H., Freeman, C. B., Gowenlock, A. H. Gut, 1972, 13, 976. 24. Starzl, T. E., Chase, H. P., Putnam, C. W., Nora, J. J. Lancet, 1974, ii, 714. 25. Starzl, T. E., Chase, H. P., Putnam, C. W., Nora, J. J., Fennell, R. H., Porter, K. A. ibid. p. 1263. 26. Grundy, S. M., Ahrens, E. H., Jr., Salen, G., Schreibman, P. H., Nestel, P. J. J. Lipid Res. 1972, 13, 531. 27. Sodhi, H. S., Kudchodkar, B. J., Horlick, L. Atherosclerosis, 1973,
17, 1. 28. 29.
Jagannathan, S. N., Connor, W. E., Baker, W. H., Bhattacharyya, A. K. J. clin. Invest. 1974, 54, 366. Miller, G. J., Miller, N. E. Lancet, 1975, i, 16.
