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BEZAFIBRATE LOWERS OXYGEN AFFINITY OF HAEMOGLOBIN
881
Preliminary Communication BEZAFIBRATE LOWERS OXYGEN AFFINITY OF HAEMOGLOBIN C. POYART
M. F. PERUTZ
MRC Laboratory of Molecular Biology, Cambridge; and INSERM, 92150 Suresnes, France
IN a search for possible drugs against sickle cell anaemia, Abraham et all found that clofibric acid has anti-sickling properties and shifts the oxygen equilibrium curve of normal human haemoglobin (Hb) to the right.’ This led us to another examine antihyperlipoproteinaemia drug, bezafibrate (’Bezalip’). Its effect on the solubility of sickle cell Hb is opposite to that of clofibric acid, so that it would enhance sickling, but it shifts the oxygen equilibrium in the same direction. In fact it lowers the oxygen affinity of normal human Hb more strongly than does the natural allosteric effector 2,3-diphosphoglycerate (2,3-DPG), and it combines with different binding sites in the Hb molecule. Unlike organic phosphates that lower the oxygen affinity, bezafibrate passes freely into and out of the red blood cell, and it does not appear to be bound by serum albumin. These properties may have clinical applications. RESULTS
Fig 1 and the table show the influence of 5 mmol/1 bezafibrate on the oxygen equilibrium curve of a solution of HbA at pH 7 - 2. The P 50 is raised from 5 to 19 mm Hg, while Hill’s coefficient remains virtually unchanged. Bezafibrate and 2,3-DPG enhance each other’s effect on the curve: while 5 mmol/1 bezafibrate raises the P50 by 14 mm Hg and 10 mmol/1 2,3-DPG raises it by 9 mm Hg, the two effectors combined raise the P50 by 33 mm Hg. The effect of bezafibrate on P50 is nearly three times larger than that of clofibric acid. Fig 2 shows the effect of bezafibrate on normal human erythrocytes suspended in isotonic buffer solution ofpH 7 - 4, containing 3 g/dl bovine serum albumin. 40 min incubation with 5 mmol/1 bezafibrate raises P 50 from 25-0to 37-66
5
Pittman M, Furman BL, Wardlaw AC. Bordetella pertussis respiratory tract infection in the mouse: pathophysiological responses. J Infect Dis 1980; 142: 56-65.
Y, Izumiya K, Sato H, Cowell JL, Manclark CR. Aerosol infection of mice with Bordetella pertussis. Infect Immun 1980; 29: 261-66. 7 Preston NW, Timewell RM, Carter EJ. Experimental pertussis infection in the rabbit: similarities with infection in primates. J Infect 1980; 2: 227-35. 8 Ashworth LAE, Fitzgeorge RB, Irons LI, Morgan CP, Robinson A. Rabbit nasopharyngeal colonization by Bordetella pertussis; the effects of immunization on clearance and on serum and nasal antibody levels. J Hyg 1982; 88: 475-86. 9 Stanbridge TN, Preston NW. Experimental pertussis infection in the marmoset: type specificity of active immunity. J Hyg 1974; 72: 213-28. 10 Ashworth LAE, Irons LI, Dowsett AB. The antigenic relationship between serotypespecific agglutinogen and fimbriae of Bordetella pertussis. Infect Immun 1982; 37: 6 Sato
11
1278-81. Irons LI, MacLennan AP. Substrate specificity and the purification by affinity combination methods of the two Bordetella pertussis haemagglutinins. In: Manclark CR, Hill JC, eds. International Symposium on Pertussis 1978. Washington DC. Department of Health Education and Welfare Publication no 79-1830, 1979: 338-49.
12 Robinson
A, Hawkins DC, Irons LI. The preparation of distinct protective antigens from Bordetella pertussis. FEMS Microbiol Lett 1981; 10: 241-44. 13 Irons LI, MacLennan AP. Isolation of the lymphocytosis promoting factorhaemagglutinin of Bordetella pertussis by affinity chromatography. Biochem Biophys Acta 1979; 580: 175-85. 14 Robinson A, Hawkins DC. Structure and biological properties ofsolubilized envelope proteins of Bordetella pertussis. Infect Immun 1983; 39: 590-98.
Fig 1-Hill plots of oxygen equilibrium curves recorded with solutions of normal human HbAo (as described by Phillips et al’). (A) control without drug or organic phosphate; (B) plus 5 mmol/I bezafibrate; (C) plus 5 mmol/I bezafibrate and 20 mmol/1 2,3-DPG. pH 7-2, 100 mmol/1 chloride, 50 mmol/1 "bis tris", 25 °C. Y =fractional saturation with oxygen. mm Hg. After washing and incubation with buffer tor another 30 min, P 50 returned to 25’66 mm Hg, showing that the drug is released from the red blood cell and that it does not damage the function of Hb. The result also shows that a EFFECTS OF ANTILIPOPROTEINAEMIA DRUGS ON OXYGEN AFFINITY OF HUMAN
*Hill’s coefficient
at
HbAo
IN ABSENCE OR PRESENCE OF
2,3-DPG
50% oxygen saturation.
t(HbA-X)isthe difference between P50 in the presence ofdrug (with or without 2,3-DPG) and P 50 for the control experiment. Bezafibrate 2-[4-(2--chlorobenzamidoethyt) phenoxy]-2-methylpropionic acid. Clofibric acid 2-(4-chlorophenoxy)-2-methylpropionate. =
15. MacLennan AP.
Specific Lipopolysaccharides of Bordetella. Biochem J 1960;
74:
398-409.
PK, Kawaoi A. Peroxidase-labelled antibody. A new method of conjugation. J Histochem Cytochem 1974; 22: 1084-91. 17. Irons LI, Ashworth LAE, Wilton-Smith P. Heterogeneity of the filamentous haemagglutinin of Bordetella pertussis studied with monoclonal antibodies. J Gen Microbiol (in press). 18. Solomn JB. Foetal and neonatal immunology. North Holland Frontiers of Biology, Neuberger A, Tatum EL, eds Amsterdam/London: North Holland publishing company. Vol 20, 1971. 19. Arbuthnott JP, Smyth CJ. Bacterial adhesion in host/pathogen interactions in animals. Ellwood DC, Melling J, Rutter P, eds. Adhesion of microorganisms to surfaces. Society for General Microbiology. New York: Academic Press, 1979: 165-98. 20. Sato Y, Izumiya K, Sato H, Cowell JL, Manclark CR. Role of antibody to leukocytosispromoting factor haemagglutinin and to filamentous haemagglutinin in immunity to pertussis. Infect Immun 1981; 31: 1223-31. 21. Dodd DC, Eisenstem BI. Antigenic quantitation of type 1 fimbriae on the surface of Escherichia coli cells by an enzyme-linked immunosorbent inhibition assay. Infect Immun 1982; 38: 764-73. 22. Bernier RH. Prospects for a new pertussis vaccine. 17th Immunization Conference Proceedings, Atlanta, Georgia: US Dept Health, 1982: 77-78. 23. Bhandari B, Pamecha RK, Mandowara SL. Seroconversion following primary immunization with D.P.T. vaccine: two versus three doses. Indian Paediatr 1981; 16. Nakane
18: 41-47. 24. Mertsola
J, Ruuskanen O, Kuronen T, Viljanen MK. Serological diagnosis of Pertussis: comparison of enzyme-linked immunosorbent assay and bacterial agglutination. J Infect Dis 1983; 147: 252-57.
882
Fig 3-Oxygen equilibrium
curves.
(A) fresh red blood cells from patient heterozygous for a Hb with very high oxygen affinity (Asp 99p"*Val); (B) curve for normal fresh red blood cells, (C) cells from the same patient as in (A) after 30 min incubation with 5 mmol/l bezafibrate at 37°C. The right shift is greatest in the physiologically important, upper part of the curve. Bezafibrate lowers the oxygen saturation at the mixed venous point (40 mm Hg) from 85% to 72%, thus doubling the amount of oxygen that can be delivered for the same difference of PO:, indicated by the arrows.
Fig 2-Hill plot of oxygen equilibrium curves recorded suspension of fresh human red cells from normal donor.
with
(A) control without drug; (B) after 40 min incubation at 37°C in an isotomc buffer containing 5 mmol/1 bezafibrate; (C) cells incubated with bezafibrate as in (B), after washing and incubation with drug-free isotonic buffer at 37°C for 30 min. pH 7-40, 140 mmol/1 sodium chloride, 10 mmol/1 glucose, 37°C.
concentration of serum albumin does not with the drug’s influence on the oxygen equilibrium. When normal human blood was incubated with 5 mmol/1 bezafibrate for 40 min Pso was raised from 24 to 33
physiological interfere
binds to a pair of sites betweeen the two a-chains in the central cavity of the Hb molecule (M. F. P., D. J. Abraham, B. Luisi, C. P., unpublished). This implies that it would lower the oxygen affinity of fetal human Hb to a degree similar to that of the adult. It is not yet clear whether a concentration of bezafibrate sufficient to benefit the oxygen equilibrium can be built up in the red cells without the administration of doses that would prove toxic. Correspondence should be addressed to Biology, Cambridge CB2 2QH.
DISCUSSION
To
M. F. P., MRC
Laboratory
of
Molecular
mm Hg.
REFERENCES
knowledge this is the first time that it has become possible to manipulate the function ofHb in the red blood cell effectively by a commonly used drug. This may have applications in several areas of clinical medicine. In patients whose polycythaemia is either idiopathic or due to the presence of an abnormal Hb with high oxygen affinity, a right shift of the curve would enhance oxygen delivery. Fig 3 our
shows the effect of bezafibrate
on
the red blood cells of
a
polycythaemic patient containing about 5007o HbA and 50% of a mutant Hb with very high oxygen affinity. The lower part of the curve is dominated by the mutant Hb on which bezafibrate has no effect, while in the upper, physiologically important part of the curve, bezafibrate acts on the normal HbA, lowering its oxygen affinity enough to reduce the oxygen saturation of the red cells from 86 to 72%, a level close to that normally prevailing at the P02 in the pulmonary artery (40 mm Hg). This may reduce the synthesis of erythropoietin and thus lower the haematocrit to nearer normal levels. Other applications that come to mind are the addition of bezafibrate to stored blood to prolong the period during which a physiological oxygen affinity is maintained, or its use for improving the delivery of oxygen to ischaemic tissue, such as the myocardium after arterial obstruction of the brain. On the other hand, caution is indicated in prescribing bezafibrate to sickle cell carriers, since it lowers the oxygen affinity of HbS as much as that of HbA, and we have found that, in vitro, it enhances the polymerisation of HbS in the erythrocyte. X-ray crystallographic analysis has shown that bezafibrate
1. Abraham 2.
DJ, Perutz MF, Phillips SEV. Physiological and X-ray studies of potential antisickling agents. Proc Natl Acad Sci USA 1983; 80: 324-28. Phillips SEV, Perutz MF, Poyart C, Wajcman H. Structure and function of haemoglobin Barcelona Asp FGI(94)&bgr;→His. J Mol Biol 1983; 164: 477-80
Hypothesis HEPATITIS B SURFACE ANTIGEN COULD HARBOUR THE INFECTIVE AGENT OF AIDS MALCOLM I. MCDONALD JOHN D. HAMILTON DAVID T. DURACK Division of Infectious Diseases, Duke University Medical Center and Durham Veterans Administration Hospital, Durham, North Carolina 27710, USA
of the acquired immunodeficiency syndrome is still a mystery. The many possibilities previous!}’ (AIDS) forward range from antigen overloaduz to amyl nitrite put and from human T-cell leukaemia virus (HTLV) to use;3 African swine fever. The proposal that AIDS is in some wav related to the hepatitis B virus (HBV) is not new; neither has it been embraced with much enthusiasm. In a case-control study, only 2% (1/46) of AIDS patients had hepatitis B surface antigen (HBsAg) detectable in their sera.6 However. antibody to hepatitis B surface antigen (anti-HBs) was detected in 76% (35/46) of these patients, and core antibody alone in a further 15% (7/46). In the light of new understanding of the HBV-associated delta agent, we believe that THE
cause
Preliminary Communication BEZAFIBRATE LOWERS OXYGEN AFFINITY OF HAEMOGLOBIN C. POYART
M. F. PERUTZ
MRC Laboratory of Molecular Biology, Cambridge; and INSERM, 92150 Suresnes, France
IN a search for possible drugs against sickle cell anaemia, Abraham et all found that clofibric acid has anti-sickling properties and shifts the oxygen equilibrium curve of normal human haemoglobin (Hb) to the right.’ This led us to another examine antihyperlipoproteinaemia drug, bezafibrate (’Bezalip’). Its effect on the solubility of sickle cell Hb is opposite to that of clofibric acid, so that it would enhance sickling, but it shifts the oxygen equilibrium in the same direction. In fact it lowers the oxygen affinity of normal human Hb more strongly than does the natural allosteric effector 2,3-diphosphoglycerate (2,3-DPG), and it combines with different binding sites in the Hb molecule. Unlike organic phosphates that lower the oxygen affinity, bezafibrate passes freely into and out of the red blood cell, and it does not appear to be bound by serum albumin. These properties may have clinical applications. RESULTS
Fig 1 and the table show the influence of 5 mmol/1 bezafibrate on the oxygen equilibrium curve of a solution of HbA at pH 7 - 2. The P 50 is raised from 5 to 19 mm Hg, while Hill’s coefficient remains virtually unchanged. Bezafibrate and 2,3-DPG enhance each other’s effect on the curve: while 5 mmol/1 bezafibrate raises the P50 by 14 mm Hg and 10 mmol/1 2,3-DPG raises it by 9 mm Hg, the two effectors combined raise the P50 by 33 mm Hg. The effect of bezafibrate on P50 is nearly three times larger than that of clofibric acid. Fig 2 shows the effect of bezafibrate on normal human erythrocytes suspended in isotonic buffer solution ofpH 7 - 4, containing 3 g/dl bovine serum albumin. 40 min incubation with 5 mmol/1 bezafibrate raises P 50 from 25-0to 37-66
5
Pittman M, Furman BL, Wardlaw AC. Bordetella pertussis respiratory tract infection in the mouse: pathophysiological responses. J Infect Dis 1980; 142: 56-65.
Y, Izumiya K, Sato H, Cowell JL, Manclark CR. Aerosol infection of mice with Bordetella pertussis. Infect Immun 1980; 29: 261-66. 7 Preston NW, Timewell RM, Carter EJ. Experimental pertussis infection in the rabbit: similarities with infection in primates. J Infect 1980; 2: 227-35. 8 Ashworth LAE, Fitzgeorge RB, Irons LI, Morgan CP, Robinson A. Rabbit nasopharyngeal colonization by Bordetella pertussis; the effects of immunization on clearance and on serum and nasal antibody levels. J Hyg 1982; 88: 475-86. 9 Stanbridge TN, Preston NW. Experimental pertussis infection in the marmoset: type specificity of active immunity. J Hyg 1974; 72: 213-28. 10 Ashworth LAE, Irons LI, Dowsett AB. The antigenic relationship between serotypespecific agglutinogen and fimbriae of Bordetella pertussis. Infect Immun 1982; 37: 6 Sato
11
1278-81. Irons LI, MacLennan AP. Substrate specificity and the purification by affinity combination methods of the two Bordetella pertussis haemagglutinins. In: Manclark CR, Hill JC, eds. International Symposium on Pertussis 1978. Washington DC. Department of Health Education and Welfare Publication no 79-1830, 1979: 338-49.
12 Robinson
A, Hawkins DC, Irons LI. The preparation of distinct protective antigens from Bordetella pertussis. FEMS Microbiol Lett 1981; 10: 241-44. 13 Irons LI, MacLennan AP. Isolation of the lymphocytosis promoting factorhaemagglutinin of Bordetella pertussis by affinity chromatography. Biochem Biophys Acta 1979; 580: 175-85. 14 Robinson A, Hawkins DC. Structure and biological properties ofsolubilized envelope proteins of Bordetella pertussis. Infect Immun 1983; 39: 590-98.
Fig 1-Hill plots of oxygen equilibrium curves recorded with solutions of normal human HbAo (as described by Phillips et al’). (A) control without drug or organic phosphate; (B) plus 5 mmol/I bezafibrate; (C) plus 5 mmol/I bezafibrate and 20 mmol/1 2,3-DPG. pH 7-2, 100 mmol/1 chloride, 50 mmol/1 "bis tris", 25 °C. Y =fractional saturation with oxygen. mm Hg. After washing and incubation with buffer tor another 30 min, P 50 returned to 25’66 mm Hg, showing that the drug is released from the red blood cell and that it does not damage the function of Hb. The result also shows that a EFFECTS OF ANTILIPOPROTEINAEMIA DRUGS ON OXYGEN AFFINITY OF HUMAN
*Hill’s coefficient
at
HbAo
IN ABSENCE OR PRESENCE OF
2,3-DPG
50% oxygen saturation.
t(HbA-X)isthe difference between P50 in the presence ofdrug (with or without 2,3-DPG) and P 50 for the control experiment. Bezafibrate 2-[4-(2--chlorobenzamidoethyt) phenoxy]-2-methylpropionic acid. Clofibric acid 2-(4-chlorophenoxy)-2-methylpropionate. =
15. MacLennan AP.
Specific Lipopolysaccharides of Bordetella. Biochem J 1960;
74:
398-409.
PK, Kawaoi A. Peroxidase-labelled antibody. A new method of conjugation. J Histochem Cytochem 1974; 22: 1084-91. 17. Irons LI, Ashworth LAE, Wilton-Smith P. Heterogeneity of the filamentous haemagglutinin of Bordetella pertussis studied with monoclonal antibodies. J Gen Microbiol (in press). 18. Solomn JB. Foetal and neonatal immunology. North Holland Frontiers of Biology, Neuberger A, Tatum EL, eds Amsterdam/London: North Holland publishing company. Vol 20, 1971. 19. Arbuthnott JP, Smyth CJ. Bacterial adhesion in host/pathogen interactions in animals. Ellwood DC, Melling J, Rutter P, eds. Adhesion of microorganisms to surfaces. Society for General Microbiology. New York: Academic Press, 1979: 165-98. 20. Sato Y, Izumiya K, Sato H, Cowell JL, Manclark CR. Role of antibody to leukocytosispromoting factor haemagglutinin and to filamentous haemagglutinin in immunity to pertussis. Infect Immun 1981; 31: 1223-31. 21. Dodd DC, Eisenstem BI. Antigenic quantitation of type 1 fimbriae on the surface of Escherichia coli cells by an enzyme-linked immunosorbent inhibition assay. Infect Immun 1982; 38: 764-73. 22. Bernier RH. Prospects for a new pertussis vaccine. 17th Immunization Conference Proceedings, Atlanta, Georgia: US Dept Health, 1982: 77-78. 23. Bhandari B, Pamecha RK, Mandowara SL. Seroconversion following primary immunization with D.P.T. vaccine: two versus three doses. Indian Paediatr 1981; 16. Nakane
18: 41-47. 24. Mertsola
J, Ruuskanen O, Kuronen T, Viljanen MK. Serological diagnosis of Pertussis: comparison of enzyme-linked immunosorbent assay and bacterial agglutination. J Infect Dis 1983; 147: 252-57.
882
Fig 3-Oxygen equilibrium
curves.
(A) fresh red blood cells from patient heterozygous for a Hb with very high oxygen affinity (Asp 99p"*Val); (B) curve for normal fresh red blood cells, (C) cells from the same patient as in (A) after 30 min incubation with 5 mmol/l bezafibrate at 37°C. The right shift is greatest in the physiologically important, upper part of the curve. Bezafibrate lowers the oxygen saturation at the mixed venous point (40 mm Hg) from 85% to 72%, thus doubling the amount of oxygen that can be delivered for the same difference of PO:, indicated by the arrows.
Fig 2-Hill plot of oxygen equilibrium curves recorded suspension of fresh human red cells from normal donor.
with
(A) control without drug; (B) after 40 min incubation at 37°C in an isotomc buffer containing 5 mmol/1 bezafibrate; (C) cells incubated with bezafibrate as in (B), after washing and incubation with drug-free isotonic buffer at 37°C for 30 min. pH 7-40, 140 mmol/1 sodium chloride, 10 mmol/1 glucose, 37°C.
concentration of serum albumin does not with the drug’s influence on the oxygen equilibrium. When normal human blood was incubated with 5 mmol/1 bezafibrate for 40 min Pso was raised from 24 to 33
physiological interfere
binds to a pair of sites betweeen the two a-chains in the central cavity of the Hb molecule (M. F. P., D. J. Abraham, B. Luisi, C. P., unpublished). This implies that it would lower the oxygen affinity of fetal human Hb to a degree similar to that of the adult. It is not yet clear whether a concentration of bezafibrate sufficient to benefit the oxygen equilibrium can be built up in the red cells without the administration of doses that would prove toxic. Correspondence should be addressed to Biology, Cambridge CB2 2QH.
DISCUSSION
To
M. F. P., MRC
Laboratory
of
Molecular
mm Hg.
REFERENCES
knowledge this is the first time that it has become possible to manipulate the function ofHb in the red blood cell effectively by a commonly used drug. This may have applications in several areas of clinical medicine. In patients whose polycythaemia is either idiopathic or due to the presence of an abnormal Hb with high oxygen affinity, a right shift of the curve would enhance oxygen delivery. Fig 3 our
shows the effect of bezafibrate
on
the red blood cells of
a
polycythaemic patient containing about 5007o HbA and 50% of a mutant Hb with very high oxygen affinity. The lower part of the curve is dominated by the mutant Hb on which bezafibrate has no effect, while in the upper, physiologically important part of the curve, bezafibrate acts on the normal HbA, lowering its oxygen affinity enough to reduce the oxygen saturation of the red cells from 86 to 72%, a level close to that normally prevailing at the P02 in the pulmonary artery (40 mm Hg). This may reduce the synthesis of erythropoietin and thus lower the haematocrit to nearer normal levels. Other applications that come to mind are the addition of bezafibrate to stored blood to prolong the period during which a physiological oxygen affinity is maintained, or its use for improving the delivery of oxygen to ischaemic tissue, such as the myocardium after arterial obstruction of the brain. On the other hand, caution is indicated in prescribing bezafibrate to sickle cell carriers, since it lowers the oxygen affinity of HbS as much as that of HbA, and we have found that, in vitro, it enhances the polymerisation of HbS in the erythrocyte. X-ray crystallographic analysis has shown that bezafibrate
1. Abraham 2.
DJ, Perutz MF, Phillips SEV. Physiological and X-ray studies of potential antisickling agents. Proc Natl Acad Sci USA 1983; 80: 324-28. Phillips SEV, Perutz MF, Poyart C, Wajcman H. Structure and function of haemoglobin Barcelona Asp FGI(94)&bgr;→His. J Mol Biol 1983; 164: 477-80
Hypothesis HEPATITIS B SURFACE ANTIGEN COULD HARBOUR THE INFECTIVE AGENT OF AIDS MALCOLM I. MCDONALD JOHN D. HAMILTON DAVID T. DURACK Division of Infectious Diseases, Duke University Medical Center and Durham Veterans Administration Hospital, Durham, North Carolina 27710, USA
of the acquired immunodeficiency syndrome is still a mystery. The many possibilities previous!}’ (AIDS) forward range from antigen overloaduz to amyl nitrite put and from human T-cell leukaemia virus (HTLV) to use;3 African swine fever. The proposal that AIDS is in some wav related to the hepatitis B virus (HBV) is not new; neither has it been embraced with much enthusiasm. In a case-control study, only 2% (1/46) of AIDS patients had hepatitis B surface antigen (HBsAg) detectable in their sera.6 However. antibody to hepatitis B surface antigen (anti-HBs) was detected in 76% (35/46) of these patients, and core antibody alone in a further 15% (7/46). In the light of new understanding of the HBV-associated delta agent, we believe that THE
cause