- Email: [email protected]
EFFECT OF BW12C ON OXYGEN AFFINITY OF HAEMOGLOBIN IN SICKLE-CELL DISEASE
831
deficiency
were
aged
under 2 years. Furthermore, the age-
normal range was calculated from a sufficiently large number of healthy children (31 aged 7-11 months and 42 aged 12-23 months) and after logarithmic transformation of the data. In the severe IgA deficiency group most children (31/40) had undectectable serum IgA (<0 - 5 mg/dl) and no secretory IgA in unstimulated saliva for the whole follow-up period; only 9 had serum IgA between 0 - 5 mg/dl and 5 mg/dl and undetectable secretory IgA. Buckley et al8 also found that severe IgA deficiency persisted in practically all children (68/70) over a 5-year period. However, Ostergaard6 found that serum IgA rose to normal in 33% of 18 children with IgA deficiency (<1 mg/dl); in his patients secretory IgA was always undetectable (<0’ 7 mg/dl) at the time of diagnosis, but there is no indication as to whether the assay was done on stimulated or unstimulated saliva. We have found that assay of secretory IgA in saliva is more reliable when done on unstimulated saliva, because there is a variable increase in and hence a diluting effect, when salivary secretion is stimulated. 13 In 20 of the 40 patients with partial IgA deficiency in our study, serum IgA levels had been spontaneously restored to normal by a median age of 14 years; this value is obtained on the assumption that the IgA deficiency was present from birth and was always partial. Although an initial severe IgA deficiency cannot be excluded, it seems unlikely because both our study and others8 suggest that severe IgA deficiency is generally persistent. However, there is the possibility that IgA levels started off normal and fell shortly before presentation with frequent infections, atopic disorders, or immunomediated diseases. Such a possibility would mean that half of the patients with partial IgA deficiency had serum IgA levels restored to normal within 4 years of diagnosis and by 14 years of age. Finally our finding that pneumonia occurs more frequently in patients with severe than with partial IgA deficiency and that restoration of IgA levels to normal is limited to children with partial IgA deficiency should be interpreted with caution because the subjects in this study had all presented with clinical symptoms and therefore may be only partly representative of the whole IgA deficient
salivary volume,
population. We thank Mr A. Ascione for technical assistance. This work was partly supported by Centro Nazionale delle Ricerche, Rome, Italy. Correspondence should be addressed to A. P., Clinica Pediatrica, Universita di Pavia, 27100 Pavia, Italy.
EFFECT OF BW12C ON OXYGEN AFFINITY OF HAEMOGLOBIN IN SICKLE-CELL DISEASE A. J. KEIDAN R. D. WHITE E. R. HUEHNS
I. M. FRANKLIN
M. JOY J. STUART Departments of Haematology, University of Birmingham and University College Hospital, London; and Wellcome Research Laboratories, Beckeriham
Eight subjects with sickle-cell disease in the symptom-free steady-state received a single one-hour infusion of the new anti-sickling agent BW12C on a total of eleven occasions. A dose-dependent increase in wholeblood oxygen affinity was observed, resulting from the action of BW12C in stabilising the oxy-conformation of haemoglobin and causing a left shift of the oxygen saturation curve. At the highest dose given (20 mg/kg bodyweight), up to 23% of haemoglobin was modified to a BW12C-reacted high-
Summary
affinity form without evidence of tissue hypoxia. There was biochemical and rheological evidence for a transient decrease in
haemolytic rate. Introduction
SICKLE-CELL disease is caused by the homozygous inheritof the abnormal haemoglobin (Hb) S ((36, Glu-Val). When deoxygenated, HbS forms rigid polymers inside the erythrocyte causing a loss of deformability which leads to the haemolytic anaemia, vaso-occlusive (sickle) crises, and other clinical features of sickle-cell disease.’,’ Deoxy-HbS is essential for the formation of polymer, and the presence of oxy-HbS inhibits polymerisation. Experimental data suggest that the maintenance of 25-30% HbS in the oxy(R)-conformation at venous oxygen tension (PO2) would prevent sickling.3 BW12C (5-[2-formyl-3-hydroxyphenoxyl pentanoic acid) was designed to bind to and stabilise the oxy-conformation of haemoglobin, thus increasing the oxygen affinity and causing a left-shift of the oxygen saturation curve.4 In healthy volunteers (homozygous HbA) a 60-minute infusion of BW12C caused a dose-dependent left-shift of the oxygen saturation curve without adverse systemic effects. The compound has anti-sickling activity in vitro, inhibiting the formation of reversibly-sickled cells4 and preserving the filterability of sickle-cells through 5 m diameter pores. This is the first report of the administration of this type of agent to patients with sickle-cell disease. ance
REFERENCES 1 Bachman R. Studies
2
&ggr;-A-globulin level. III. The frequency of a-&ggr;-Aglobulinemia. Scand J Clin Lab Invest 1965; 17: 316-20. Johansson SGO, Hogman CF, Killander J. Quantitative immunoglobulin on
the
serum
determination. Acta Pathol Microbiol Scand 1968; 74: 519-30. Buckley RH, Dees SC. Correlation of milk precipitins with IgA deficiency. N EnglJ Med 1969; 218: 465-69. 4 Amman AJ, Hong R. Selective IgA deficiency: Presentation of 30 cases and a review of the literature. Medicine 1971; 50: 223-36. 5. Ugazio AG, Out TA, Plebani A, et al. Recurrent infections in children with "selective" IgA deficiency: Association with IgG2 and IgG4 deficiency. In: Wedgwood, GJ, Rosen FS, Paul NW (eds). Primary immunodeficiency diseases. Liss AR, and March of Dimes Birth Defects Foundation, New York: 1983: 169-71. 6. Ostergaard PA. Clinical and immunological features of transient IgA deficiency in children. Clin Exp Immunol 1980; 40: 561-64. 7. Savilahti E, Pelkonen P. Clinical findings and intestinal immunoglobulins in children with partial IgA deficiency. Acta Paediatr Scand 1979; 68: 513-19. 8. Buckley RH. Clinical and immunologic features of selective IgA deficiency. In: Bergsma D, ed. Immunodeficiency in man and animals. Sunderland, Mass: Sinauer Associates, 1975: 134-41. 9. Johns P. Felix-Davies DD, Hawkins CF, et al. IgA deficiency in patients with rheumatoid arthritis treated with D-penicillamine or gold. Ann Rheum Dis 1978; 37: 289. 3
Seager J, Jamison DL, Wilson J, Hayward AR, Soothill JF. IgA deficiency, epilepsy, and phenytoin treatment. Lancet 1975; ii: 632-35. 11. Mancini G, Carbonara AO, Heremans JF. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 1965; 2: 235-54. 12. Plebani A, Mira E, Mevio E, et al. IgM and IgD concentrations in the serum and secretions of children with selective IgA deficiency. Clin Exp Immunol 1983; 53: 10.
689-96.
13.
Burgio GR, Lanzavecchia A, Plebani A, Jayakar S, Ugazio AG. Ontogeny of secretory immunity: Levels of secretory IgA and natural antibodies in saliva. Pediatr Res 1980;
14: 1111-14. 14. Tomasi TB, Grey HM. Structure and function of immunoglobulin A. Prog Allergy 1972; 16: 81-213. 15. Gehan EA. Statistical methods for survival time studies. In: Staquet MJ (ed). Cancer therapy: Prognostic factors and criteria of response. New York: Raven Press, 1975: 7-35. 16. Tomasi TB, Tau EM, Solomon A, Prendergast RA. Characteristics of an immune system common to certain external secretions. J Exp Med 1965; 121: 101-24. 17. Isaacs D, Altman DG, Tidmarsh CE, Valman HB, Webster ADB. Serum immunoglobulin concentrations in preschool children measured by laser nephelometry: reference ranges for IgG, IgA, IgM. J Clin Parhol 1983; 36: 1193-96.
832
Patients and Methods
Eight otherwise healthy men of mean age 22 -6yr (range 19 - 34) with homozygous sickle-cell disease or sickle-&bgr;o thalassaemia were recruited and gave their informed (HbA 0%; HbF<10%) consent to the investigation. Three were studied on two occasions (table I). None of the subjects had had a painful crisis in the two weeks before testing. A clinical trial exemption certificate was granted by the Committee on Safety of Medicines and the study was approved by the research ethical committees of the Central Birmingham Health Authority and of University College Hospital, London. Fasting venous blood samples were taken and the required dose of BW1 2C, diluted in 20 ml saline, was infused over 60 minutes into an antecubital vein; the initial seven doses were adjusted for patient haematocrit. Venous blood samples were taken from the opposite arm immediately postinfusion and 1, 2, 4, 8, 12, 24, and in some cases 48 h after the infusion. The subjects remained in bed during the infusion and for 6 h afterwards, and their clinical status, continuous electrocardiogram, and fluid balance were monitored. They remained in hospital under observation for 24 h after the infusion. Coulter S profile (Coulter Electronics Ltd, Luton), manual white cell differential, and reticulocyte and nucleated red cell counts were obtained by standard techniques.Red cell morphology was examined by interference microscopy of wet-mount slides of fixed cells prepared by collection of 1 ml venous blood into 9 ml 2’ 5% w/v glutaraldehyde in phosphate buffered saline (PBS, 75 mmol/1 phosphate) previously equilibrated with 5% 02/95% N2 to give a P02 of approximately 4 - 8 kPa (1kPa =7-55 mm Hg). 500 cells were counted and classified into sickled cells, spiculated cells, bizarre cells, and discocytes.8 An additional sample of oxygenated venous blood was fixed for irreversibly sickled cell count. Biochemical analyses were performed by standard methods. Freeflowing venous blood lactate and whole blood 2,3-diphosphoglycerate (2,3-DPG) concentrations were measured enzymatically. The whole-blood oxygen saturation curve was measured with a ’Hem-O-Scan’ (Aminco, Silver Springs, Maryland, USA). Three values were determined from each association curve-P50, the oxygen tension (kPa) at which haemoglobin is 50% saturated with oxygen; P20, the POl at 20% saturation; and S10, the percentage oxygen saturation at a P02 of 1’3kPa (10 mm Hg) (table I). A maximally left-shifted oxygen saturation curve (P50 0,99 kPa, P20 0 -3kPa, S 10 65’5%) was produced by incubation of a preinfusion blood sample with 15 mmol/1 BW12C at room temperature.4Each postinfusion saturation curve was analysed by comparison with a series of artificially constructed curves (templates) calculated from the equation of Bed dell et a1.4 Templates were prepared for a range of P50 values and proportion of BW12C-reacted Hb, on the assumption that two Hb species were present-unreacted HbS and haemoglobin fully reacted with BW12C (fig 1). The proportion of BW12C-reacted haemoglobin (%MOD) was determined from the artificial curve that exactly corresponded to the observed postinfusion curve.
TABLE I-EFFECTS OF BW
*t:Studied on two occasions.
12c ON OXYGEN SATURATION
CURVES
Fig 1-Artificially constructed (template) curves showing relation between oxygen tension (kPa) and oxygen saturation (%) for varying proportions of BW12C-reacted haemoglobin. In this
example, the P50 of the control curve is 5 - 5
kPa.
Erythrocyte deformability was measured at ambient temperature the index of filtration (IF) of pure erythrocyte suspensions in PBS (haematocrit 0-04-0-05) as determined by an initial-flow-rate filtration method (’Hémorhéomètre’; 5 Pm pore diameter polycarbonate membranes).9 An increase in IF indicates loss of erythrocyte filterability (deformability). Red cell suspensions were prepared for filtration as previously describedl° and deoxygenated for varying periods with a 5% C02/95% N2 gas mixture before as
simultaneous measurement of IF and P02. The critical P02 for filtration, at which IF became double the value of a fully oxygenated 1
sample, was determined." Concentrations
of BW12C in plasma and whole blood .5 measured as previously described Pharmacokinetic analysis of drug levels was performed at Wellcome Research Laboratories. Statistical significance (two-tail) was determined by Wilcoxon’s signed rank test for paired data.
haemolysates
were
Results
No change in the clinical condition of the subjects was noted during the observation period. The BW12C infusion caused local venous irritation but this was prevented by selection of a larger vein and infusion of the drug in parallel with 100-200 ml saline through a Y-connector. Oxygen saturation curves were shifted to the left in all subjects after the infusion (fig 2, table l). As in the normal volunteers,5postinfusion saturation curves were biphasic, and were assumed to represent the proportional summation of curves for unreacted (preinfusion) haemoglobin and haemoglobin fully reacted with BW12C. The %MOD, calculated on this assumption, correlated with the administered dose of BW12C (r=0-88; p<0’001). The highest %MOD was 23% (table I). The left-shift was chiefly manifest at the lower end of the oxygen saturation curve, as seen in normal volunteers,5 but in five subjects the whole curve was left-shifted to some degree. Return of the saturation curve to preinfusion values was variable: three subjects (3, 8, 11) showed a persistent left-shift in P20 at 24 h and three (5, 7, 9) had an overswing of P50 to the right at 24 h although BW12C could no longer be detected in the blood. No change in 2,3-DPG was observed in the six subjects in whom this was measured. Pharmacokinetic studies confirmed the high specificity of BW12C binding to haemoglobin in vivo, since the largest
833 serum
concentrations of total bilirubin and aspartate, but not
alanine, aminotransferase were significantly lower than the preinfusion values (p<0 02 and =0’002, respectively) (table II); a parallel fall in serum hydroxybutyrate dehydrogenase was not quite statistically significant at the 5% level. Nothing was observed to suggest any detrimental effect of the drug. No significant trends suggestive of tissue hypoxia were observed in venous P02, blood lactate, or creatine kinase during the 24 h following the infusion. Erythrocyte filterability (IF) did not improve after BW12C, and at 4 h postinfusion five of seven subjects studied had an increase in IF (table III). The critical P02 for change in the postinfusion period.
filtration showed little
Discussion Fig 2-Preinfusion (i) and postinfusion (ii) oxygen saturation curves for infusion no 11, and fully reacted oxygen saturation curve (iii). Oxygen saturation (%) is plotted against oxygen tension (kPa).
proportion of the dose was found in the erythrocytes (mean erythrocyte:plasma ratio 14, SEM 0 - 98). Initial half-life was 1-66±0-38 h and late half-life 9-26±3-05 h. The wholeblood concentration at the end of the infusion was highly dose-dependent and showed a linear relation (r=0’99, p<0 001). There was no significant effect of dose on the halflives of elimination of B W 12C. No significant trends in reticulocyte, nucleated red cell, or irreversibly sickled cell counts were observed. At 4 h the TABLE II-BIOCHEMICAL INDICES BEFORE AND -
INFUSION
4 HOURS AFTER
OF BW 12c
TABLE III-ERYTHROCYTE DEFORMABILITY BEFORE AND AFTER
INFUSION OF BW 12c
The morbidity and mortality in sickle-cell disease, both in the UK12 and in Jamaica,l3 can be attributed mainly to the acute and chronic complications of microvascular occlusion by rheologically compromised erythrocytes. 14 The intracellular concentration of deoxy-HbS is the critical determinant of haemoglobin polymerisation and hence sickling.l Agents previously used to increase the proportion of liganded HbS in vivo have included carbon monoxide, sodium nitrite, and cyanate.1’ Cyanate inhibited sickling in vitro but could not be used in treatment because of toxicity and lack of specificity for haemoglobin. 16 BW12C is the most potent of a series of substituted benzaldehydes specifically designed to increase the oxygen affinity of haemoglobin and inhibit sickling.4 The drug binds preferentially to the oxy-conformation of haemoglobin at a site between the amino-terminal residues of the a-subunits, thus stabilising the oxy-conformation and causing a dosedependent left-shift of the oxygen saturation curve. The effect of BW12C on haemoglobin is essentially stoicheiometric and the resultant oxygen saturation curve is biphasic, being a composite of curves for two functionally different haemoglobin forms with different oxygen affinities: this allows the percentage of BW12C-modified, high-affinity 4 haemoglobin (%MOD) to be determined.4 In sickle-cell disease the oxygen saturation curve is complicated by the presence of intracellular polymer and the preinfusion curve may not be the appropriate "template" for the unreacted form of haemoglobin as required in the above analysis: for this reason artificially constructed curves were used in determination of %MOD. At BW12C doses of 20 mg/kg, up to 23% of the haemoglobin was modified to a high-affinity form, approaching levels of theoretical clinical benefit in sickle-cell disease.3 Modified haemoglobin is unavailable for oxygen transport, but no clinical or biochemical evidence of tissue
hypoxia was seen. Despite the documented anti-sickling activity ofBW 12C in vitro,4,6 it would be surprising to see a reduction in the haemolytic rate over the short time-scale of this study. Interpretation of the biochemical changes is hampered by the lack of a control group on bed rest alone. However, the small falls in serum bilirubin and aspartate aminotransferase concentrations do indicate a reduced rate ofhaemolysis in the immediate postinfusion period. Preservation of rheologically compromised erythrocytes could explain the impairment in erythrocyte filtrability observed at 4 h; in-vitro studies have demonstrated the high sensitivity of IF to increasing percentages of irreversibly sickled cells added to sickle whole blood These observations suggest that short-term
834
infusions of BW12C should be tailed off to prevent any rebound increase in sickling caused by the temporarily longer survival of rheologically compromised cells. An analogue with a longer half-life might overcome this difficulty. Progressive deoxygenation of sickle blood in vitro hampers filtration of erythrocytes under positive pressure through 5 m diameter pores. This impairment can be prevented by the in-vitro addition of BW12C concentrations ofl’5mmol/1 and above.6 Similarly, the initial-flow-rate gravity filtration method used in the present study showed a progressive fall in the critical P02 for filtration when BW12C was added in increasing concentration (0-3 mmol/1) to sickle blood invitro. Only one of six subjects, however, showed a fall in the critical P02 at a BW12C concentration off 75 mmol/1 (the maximum whole blood concentration achieved in vivo with intravenous infusion). This result, and the apparent absence of rheological benefit in the in-vivo study, suggests that a quantitatively greater left-shift of the oxygen saturation curve may be required to secure rheological improvement in sickle cells of a degree that can be detected by our existing filtration
technique. Further studies of BW12C at higher doses for longer periods, both in the steady state and during crisis, will be ’needed to evaluate the role of this agent in the treatment of sickle-cell disease. In these investigations, patients should be carefully monitored to ensure that "therapeutic" levels, as regards sickling, do not compromise oxygen delivery to tissues. Any reduction in oxygen delivery, in conjunction with reduced haemolysis, might cause a rise in erythrocyte mass and hence whole blood viscosity; thus rheological monitoring will be required to ensure that the rise in whole blood viscosity does not overwhelm the rheological benefit of increased
erythrocyte deformability.6
This work was supported by the Clinical and Applied Research Division of the Wellcome Foundation Ltd. A. J. K. is funded by Action Research for the Crippled Child. We are indebted to Dr M. Toop, Dr R. G. Sparks, and Mr S. S. Marwah for laboratory analyses; to Dr R. Wootton for help in the analysis of oxygen saturation curves; to Dr D. Bareford, Dr G. S. Lucas, and Dr J. Porter for patient supervision in the early stages of the study; and to the eight volunteers.
Correspondence should be addressed to J. S., Department of Haematology, University of Birmingham, Birmingham B15 2TJ.
Medical School,
REFERENCES ER. Treatment of sickle-cell disease. Trans R Soc Trop Med Hyg 1974; 68: 85-91. 2. Brittenham GM, Schechter AN, Noguchi CT. Hemoglobin S polymerization: primary determinant of the hemolytic and clinical severity of the sickling syndromes. Blood 1.
May A, Huehns
1985; 65: 183-89.
IM, Rosemeyer MA, Huehns ER. Sickle cell disease: the proportion of J 1983; 54: 579-87. liganded haemoglobin needed to prevent crises. Br Haematol 4. Beddell CR, Goodford PJ, Kneen G, White RD, Wilkinson S, Wootton R. Substituted benzaldehydes designed to increase the oxygen affinity of human haemoglobin and inhibit the sickling of sickle erythrocytes. Br J Pharmacol 1984; 82: 397-407. 5. Fitzharris P, McLean AEM, Sparks RG, Weatherley BC, White RD, Wootton R. The effects in volunteers of BW12C, a compound designed to left-shift the blood-oxygen saturation curve. Br J Clin Pharmacol 1985; 19: 471-81. 6. Kenny MW, Stuart J. Preservation of deformability (filterability) of sickle cells by BW12C during progressive deoxygenation. Br J Haematol 1983; 55: 465-71. 7. Dacie JV, Lewis SM. Practical haematology, 6th ed. Edinburgh: Churchill Livingstone, 1984. 8. Bessis M. Red cell shapes. An illustrated classification and its rationale. Nouv Rev Fr 3. Franklin
Hématol 1972; 12:
The
Management of AIDS Patients
Edited by D. Miller, J. Weber, and J. Green, St Mary’s Hospital, London. London: Macmillan. 1986. Pp 195. ,E30.
IT is hard to name a disease that has had a greater impact on in general, and the teaching hospital in particular, than AIDS. Moreover, we should try to understand why certain teaching hospitals in major cities become "AIDS central" and suffer most upheaval and readjustment. Several factors determine this phenomenon-usually the physical proximity of the institution to its AIDS risk-group clients and the expertise of the existing staff in providing service for an AIDS risk-group or for any of the clinical expressions of AIDS virus infection. St Mary’s, London, is an example of a hospital where many of these factors’have operated. In this book staff from St Mary’s outline their approach to the diagnosis and treatment of persons with AIDS and related illnesses, based on their extensive experience in venereology. For the relatively well seropositive person the venereology model works because the staff are experienced and comfortable dealing non-judgmentally with outpatient clients; the chapter written outside St Mary’s, in which haemophiliacs are seen as "innocent victims" of AIDSvirus infection, reflects a more negative attitude. When, however; the client becomes immunodeficient and requires the services of the teaching hospital department of internal medicine and its subspecialties, the venereology model is unhelpful and this book shows how effectively the changing needs of the client can be integrated in one institution. Overall, I enjoyed it. The emphasis of each chapter varies appropriately in relation to its predominant audience and the medical and scientific aspects are generally comprehensive and sound. In view of the prediction that the Centers for Disease Control surveillance definition will collapse as the dreadful prognosis of severe immune deficiency becomes accepted, I believe that the authors are overzealous in requiring a proven diagnosis of opportunistic infection in order to fulfil a rigid case definition of end-stage AIDS. Good clinical illustration is an important aspect of such a practical book. Several of the colour reproductions are unfortunate; for example, the photograph of Kaposi’s sarcoma on the buttocks also showing a tattoo is a better demonstration of British patriotism than of the characteristic appearance of the tumour, and the picture of the cotrimoxazole rash in a ventilated patient negatively reinforces an aggressive approach to management. The section on nursing aspects, emphasising total patient care, is carefully prepared and pragmatic. Although I found the patient problem analysis tables repetitive and unimaginative, the chapters on the psychosocial aspects were excellent. The suggestion that prognosis or statistics should not be discussed at initial diagnosis of full-blown AIDS strikes me as impractical as well
society
as
deceitful.
Time will tell whether the St Mary’s framework for management of AIDS virus infection is a major model or only one of many that can provide comprehensive, compassionate, and cost-effective care. I recommend this book to any professional who is involved in the care of these persons. Centre for Immunology, St Vincent’s Hospital, Sydney, NSW 2010, Australia
12.
721-46.
DAVID A. COOPER
Davis LR, Huehns ER, White JM. Survey of sickle cell disease in England and Wales Br Med J 1981; 283: 1519-21. Thomas AN, Pattison C, Serjeant GR. Causes of death in sickle cell disease in Jamaica Br Med J 1982; 285: 633-35. Stuart J. Sickle cell disease and vascular occlusion-rheological aspects. Clin Hemorheol 1984; 4: 193-207. Dean J, Schechter AN. Sickle cell anemia: molecular and cellular bases of therapeutic approaches. N Engl J Med 1978; 299: 752-65, 804-11, 863-70. Harkness DR, Roth S. Clinical evaluation of cyanate in sickle cell anemia. Prog Hematol
Erythrocyte filtrability measurement by the initial flow rate method. Biorheology 1983; 20: 199-211. Stuart J, Stone PCW, Bareford D, Caldwell NM, Davies JE, Baar S. Evaluation of leucocyte removal methods for studies oferythrocyte deformability. Clin Hemorheol
13.
1985; 5: 137-47. GS, Caldwell NM, Stuart J. Fluctuating deformability of oxygenated sickle erythrocytes in the asymptomatic state and in painful crisis. Br J Haematol 1985; 59:
15.
363-68.
1975; 9: 157-84. 17. Stuart J, Keidan AJ, Marwah SS, Green MA. Rheological methods for the evaluation of anti-sickling compounds in clinical trials. In: Beuzard Y, Charache S, Galacteros F, eds. Approaches to the therapy of sickle cell disease. INSERM Symp (in press).
9. Hanss M. 10.
Reviews of Books
14.
11. Lucas
16.
deficiency
were
aged
under 2 years. Furthermore, the age-
normal range was calculated from a sufficiently large number of healthy children (31 aged 7-11 months and 42 aged 12-23 months) and after logarithmic transformation of the data. In the severe IgA deficiency group most children (31/40) had undectectable serum IgA (<0 - 5 mg/dl) and no secretory IgA in unstimulated saliva for the whole follow-up period; only 9 had serum IgA between 0 - 5 mg/dl and 5 mg/dl and undetectable secretory IgA. Buckley et al8 also found that severe IgA deficiency persisted in practically all children (68/70) over a 5-year period. However, Ostergaard6 found that serum IgA rose to normal in 33% of 18 children with IgA deficiency (<1 mg/dl); in his patients secretory IgA was always undetectable (<0’ 7 mg/dl) at the time of diagnosis, but there is no indication as to whether the assay was done on stimulated or unstimulated saliva. We have found that assay of secretory IgA in saliva is more reliable when done on unstimulated saliva, because there is a variable increase in and hence a diluting effect, when salivary secretion is stimulated. 13 In 20 of the 40 patients with partial IgA deficiency in our study, serum IgA levels had been spontaneously restored to normal by a median age of 14 years; this value is obtained on the assumption that the IgA deficiency was present from birth and was always partial. Although an initial severe IgA deficiency cannot be excluded, it seems unlikely because both our study and others8 suggest that severe IgA deficiency is generally persistent. However, there is the possibility that IgA levels started off normal and fell shortly before presentation with frequent infections, atopic disorders, or immunomediated diseases. Such a possibility would mean that half of the patients with partial IgA deficiency had serum IgA levels restored to normal within 4 years of diagnosis and by 14 years of age. Finally our finding that pneumonia occurs more frequently in patients with severe than with partial IgA deficiency and that restoration of IgA levels to normal is limited to children with partial IgA deficiency should be interpreted with caution because the subjects in this study had all presented with clinical symptoms and therefore may be only partly representative of the whole IgA deficient
salivary volume,
population. We thank Mr A. Ascione for technical assistance. This work was partly supported by Centro Nazionale delle Ricerche, Rome, Italy. Correspondence should be addressed to A. P., Clinica Pediatrica, Universita di Pavia, 27100 Pavia, Italy.
EFFECT OF BW12C ON OXYGEN AFFINITY OF HAEMOGLOBIN IN SICKLE-CELL DISEASE A. J. KEIDAN R. D. WHITE E. R. HUEHNS
I. M. FRANKLIN
M. JOY J. STUART Departments of Haematology, University of Birmingham and University College Hospital, London; and Wellcome Research Laboratories, Beckeriham
Eight subjects with sickle-cell disease in the symptom-free steady-state received a single one-hour infusion of the new anti-sickling agent BW12C on a total of eleven occasions. A dose-dependent increase in wholeblood oxygen affinity was observed, resulting from the action of BW12C in stabilising the oxy-conformation of haemoglobin and causing a left shift of the oxygen saturation curve. At the highest dose given (20 mg/kg bodyweight), up to 23% of haemoglobin was modified to a BW12C-reacted high-
Summary
affinity form without evidence of tissue hypoxia. There was biochemical and rheological evidence for a transient decrease in
haemolytic rate. Introduction
SICKLE-CELL disease is caused by the homozygous inheritof the abnormal haemoglobin (Hb) S ((36, Glu-Val). When deoxygenated, HbS forms rigid polymers inside the erythrocyte causing a loss of deformability which leads to the haemolytic anaemia, vaso-occlusive (sickle) crises, and other clinical features of sickle-cell disease.’,’ Deoxy-HbS is essential for the formation of polymer, and the presence of oxy-HbS inhibits polymerisation. Experimental data suggest that the maintenance of 25-30% HbS in the oxy(R)-conformation at venous oxygen tension (PO2) would prevent sickling.3 BW12C (5-[2-formyl-3-hydroxyphenoxyl pentanoic acid) was designed to bind to and stabilise the oxy-conformation of haemoglobin, thus increasing the oxygen affinity and causing a left-shift of the oxygen saturation curve.4 In healthy volunteers (homozygous HbA) a 60-minute infusion of BW12C caused a dose-dependent left-shift of the oxygen saturation curve without adverse systemic effects. The compound has anti-sickling activity in vitro, inhibiting the formation of reversibly-sickled cells4 and preserving the filterability of sickle-cells through 5 m diameter pores. This is the first report of the administration of this type of agent to patients with sickle-cell disease. ance
REFERENCES 1 Bachman R. Studies
2
&ggr;-A-globulin level. III. The frequency of a-&ggr;-Aglobulinemia. Scand J Clin Lab Invest 1965; 17: 316-20. Johansson SGO, Hogman CF, Killander J. Quantitative immunoglobulin on
the
serum
determination. Acta Pathol Microbiol Scand 1968; 74: 519-30. Buckley RH, Dees SC. Correlation of milk precipitins with IgA deficiency. N EnglJ Med 1969; 218: 465-69. 4 Amman AJ, Hong R. Selective IgA deficiency: Presentation of 30 cases and a review of the literature. Medicine 1971; 50: 223-36. 5. Ugazio AG, Out TA, Plebani A, et al. Recurrent infections in children with "selective" IgA deficiency: Association with IgG2 and IgG4 deficiency. In: Wedgwood, GJ, Rosen FS, Paul NW (eds). Primary immunodeficiency diseases. Liss AR, and March of Dimes Birth Defects Foundation, New York: 1983: 169-71. 6. Ostergaard PA. Clinical and immunological features of transient IgA deficiency in children. Clin Exp Immunol 1980; 40: 561-64. 7. Savilahti E, Pelkonen P. Clinical findings and intestinal immunoglobulins in children with partial IgA deficiency. Acta Paediatr Scand 1979; 68: 513-19. 8. Buckley RH. Clinical and immunologic features of selective IgA deficiency. In: Bergsma D, ed. Immunodeficiency in man and animals. Sunderland, Mass: Sinauer Associates, 1975: 134-41. 9. Johns P. Felix-Davies DD, Hawkins CF, et al. IgA deficiency in patients with rheumatoid arthritis treated with D-penicillamine or gold. Ann Rheum Dis 1978; 37: 289. 3
Seager J, Jamison DL, Wilson J, Hayward AR, Soothill JF. IgA deficiency, epilepsy, and phenytoin treatment. Lancet 1975; ii: 632-35. 11. Mancini G, Carbonara AO, Heremans JF. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 1965; 2: 235-54. 12. Plebani A, Mira E, Mevio E, et al. IgM and IgD concentrations in the serum and secretions of children with selective IgA deficiency. Clin Exp Immunol 1983; 53: 10.
689-96.
13.
Burgio GR, Lanzavecchia A, Plebani A, Jayakar S, Ugazio AG. Ontogeny of secretory immunity: Levels of secretory IgA and natural antibodies in saliva. Pediatr Res 1980;
14: 1111-14. 14. Tomasi TB, Grey HM. Structure and function of immunoglobulin A. Prog Allergy 1972; 16: 81-213. 15. Gehan EA. Statistical methods for survival time studies. In: Staquet MJ (ed). Cancer therapy: Prognostic factors and criteria of response. New York: Raven Press, 1975: 7-35. 16. Tomasi TB, Tau EM, Solomon A, Prendergast RA. Characteristics of an immune system common to certain external secretions. J Exp Med 1965; 121: 101-24. 17. Isaacs D, Altman DG, Tidmarsh CE, Valman HB, Webster ADB. Serum immunoglobulin concentrations in preschool children measured by laser nephelometry: reference ranges for IgG, IgA, IgM. J Clin Parhol 1983; 36: 1193-96.
832
Patients and Methods
Eight otherwise healthy men of mean age 22 -6yr (range 19 - 34) with homozygous sickle-cell disease or sickle-&bgr;o thalassaemia were recruited and gave their informed (HbA 0%; HbF<10%) consent to the investigation. Three were studied on two occasions (table I). None of the subjects had had a painful crisis in the two weeks before testing. A clinical trial exemption certificate was granted by the Committee on Safety of Medicines and the study was approved by the research ethical committees of the Central Birmingham Health Authority and of University College Hospital, London. Fasting venous blood samples were taken and the required dose of BW1 2C, diluted in 20 ml saline, was infused over 60 minutes into an antecubital vein; the initial seven doses were adjusted for patient haematocrit. Venous blood samples were taken from the opposite arm immediately postinfusion and 1, 2, 4, 8, 12, 24, and in some cases 48 h after the infusion. The subjects remained in bed during the infusion and for 6 h afterwards, and their clinical status, continuous electrocardiogram, and fluid balance were monitored. They remained in hospital under observation for 24 h after the infusion. Coulter S profile (Coulter Electronics Ltd, Luton), manual white cell differential, and reticulocyte and nucleated red cell counts were obtained by standard techniques.Red cell morphology was examined by interference microscopy of wet-mount slides of fixed cells prepared by collection of 1 ml venous blood into 9 ml 2’ 5% w/v glutaraldehyde in phosphate buffered saline (PBS, 75 mmol/1 phosphate) previously equilibrated with 5% 02/95% N2 to give a P02 of approximately 4 - 8 kPa (1kPa =7-55 mm Hg). 500 cells were counted and classified into sickled cells, spiculated cells, bizarre cells, and discocytes.8 An additional sample of oxygenated venous blood was fixed for irreversibly sickled cell count. Biochemical analyses were performed by standard methods. Freeflowing venous blood lactate and whole blood 2,3-diphosphoglycerate (2,3-DPG) concentrations were measured enzymatically. The whole-blood oxygen saturation curve was measured with a ’Hem-O-Scan’ (Aminco, Silver Springs, Maryland, USA). Three values were determined from each association curve-P50, the oxygen tension (kPa) at which haemoglobin is 50% saturated with oxygen; P20, the POl at 20% saturation; and S10, the percentage oxygen saturation at a P02 of 1’3kPa (10 mm Hg) (table I). A maximally left-shifted oxygen saturation curve (P50 0,99 kPa, P20 0 -3kPa, S 10 65’5%) was produced by incubation of a preinfusion blood sample with 15 mmol/1 BW12C at room temperature.4Each postinfusion saturation curve was analysed by comparison with a series of artificially constructed curves (templates) calculated from the equation of Bed dell et a1.4 Templates were prepared for a range of P50 values and proportion of BW12C-reacted Hb, on the assumption that two Hb species were present-unreacted HbS and haemoglobin fully reacted with BW12C (fig 1). The proportion of BW12C-reacted haemoglobin (%MOD) was determined from the artificial curve that exactly corresponded to the observed postinfusion curve.
TABLE I-EFFECTS OF BW
*t:Studied on two occasions.
12c ON OXYGEN SATURATION
CURVES
Fig 1-Artificially constructed (template) curves showing relation between oxygen tension (kPa) and oxygen saturation (%) for varying proportions of BW12C-reacted haemoglobin. In this
example, the P50 of the control curve is 5 - 5
kPa.
Erythrocyte deformability was measured at ambient temperature the index of filtration (IF) of pure erythrocyte suspensions in PBS (haematocrit 0-04-0-05) as determined by an initial-flow-rate filtration method (’Hémorhéomètre’; 5 Pm pore diameter polycarbonate membranes).9 An increase in IF indicates loss of erythrocyte filterability (deformability). Red cell suspensions were prepared for filtration as previously describedl° and deoxygenated for varying periods with a 5% C02/95% N2 gas mixture before as
simultaneous measurement of IF and P02. The critical P02 for filtration, at which IF became double the value of a fully oxygenated 1
sample, was determined." Concentrations
of BW12C in plasma and whole blood .5 measured as previously described Pharmacokinetic analysis of drug levels was performed at Wellcome Research Laboratories. Statistical significance (two-tail) was determined by Wilcoxon’s signed rank test for paired data.
haemolysates
were
Results
No change in the clinical condition of the subjects was noted during the observation period. The BW12C infusion caused local venous irritation but this was prevented by selection of a larger vein and infusion of the drug in parallel with 100-200 ml saline through a Y-connector. Oxygen saturation curves were shifted to the left in all subjects after the infusion (fig 2, table l). As in the normal volunteers,5postinfusion saturation curves were biphasic, and were assumed to represent the proportional summation of curves for unreacted (preinfusion) haemoglobin and haemoglobin fully reacted with BW12C. The %MOD, calculated on this assumption, correlated with the administered dose of BW12C (r=0-88; p<0’001). The highest %MOD was 23% (table I). The left-shift was chiefly manifest at the lower end of the oxygen saturation curve, as seen in normal volunteers,5 but in five subjects the whole curve was left-shifted to some degree. Return of the saturation curve to preinfusion values was variable: three subjects (3, 8, 11) showed a persistent left-shift in P20 at 24 h and three (5, 7, 9) had an overswing of P50 to the right at 24 h although BW12C could no longer be detected in the blood. No change in 2,3-DPG was observed in the six subjects in whom this was measured. Pharmacokinetic studies confirmed the high specificity of BW12C binding to haemoglobin in vivo, since the largest
833 serum
concentrations of total bilirubin and aspartate, but not
alanine, aminotransferase were significantly lower than the preinfusion values (p<0 02 and =0’002, respectively) (table II); a parallel fall in serum hydroxybutyrate dehydrogenase was not quite statistically significant at the 5% level. Nothing was observed to suggest any detrimental effect of the drug. No significant trends suggestive of tissue hypoxia were observed in venous P02, blood lactate, or creatine kinase during the 24 h following the infusion. Erythrocyte filterability (IF) did not improve after BW12C, and at 4 h postinfusion five of seven subjects studied had an increase in IF (table III). The critical P02 for change in the postinfusion period.
filtration showed little
Discussion Fig 2-Preinfusion (i) and postinfusion (ii) oxygen saturation curves for infusion no 11, and fully reacted oxygen saturation curve (iii). Oxygen saturation (%) is plotted against oxygen tension (kPa).
proportion of the dose was found in the erythrocytes (mean erythrocyte:plasma ratio 14, SEM 0 - 98). Initial half-life was 1-66±0-38 h and late half-life 9-26±3-05 h. The wholeblood concentration at the end of the infusion was highly dose-dependent and showed a linear relation (r=0’99, p<0 001). There was no significant effect of dose on the halflives of elimination of B W 12C. No significant trends in reticulocyte, nucleated red cell, or irreversibly sickled cell counts were observed. At 4 h the TABLE II-BIOCHEMICAL INDICES BEFORE AND -
INFUSION
4 HOURS AFTER
OF BW 12c
TABLE III-ERYTHROCYTE DEFORMABILITY BEFORE AND AFTER
INFUSION OF BW 12c
The morbidity and mortality in sickle-cell disease, both in the UK12 and in Jamaica,l3 can be attributed mainly to the acute and chronic complications of microvascular occlusion by rheologically compromised erythrocytes. 14 The intracellular concentration of deoxy-HbS is the critical determinant of haemoglobin polymerisation and hence sickling.l Agents previously used to increase the proportion of liganded HbS in vivo have included carbon monoxide, sodium nitrite, and cyanate.1’ Cyanate inhibited sickling in vitro but could not be used in treatment because of toxicity and lack of specificity for haemoglobin. 16 BW12C is the most potent of a series of substituted benzaldehydes specifically designed to increase the oxygen affinity of haemoglobin and inhibit sickling.4 The drug binds preferentially to the oxy-conformation of haemoglobin at a site between the amino-terminal residues of the a-subunits, thus stabilising the oxy-conformation and causing a dosedependent left-shift of the oxygen saturation curve. The effect of BW12C on haemoglobin is essentially stoicheiometric and the resultant oxygen saturation curve is biphasic, being a composite of curves for two functionally different haemoglobin forms with different oxygen affinities: this allows the percentage of BW12C-modified, high-affinity 4 haemoglobin (%MOD) to be determined.4 In sickle-cell disease the oxygen saturation curve is complicated by the presence of intracellular polymer and the preinfusion curve may not be the appropriate "template" for the unreacted form of haemoglobin as required in the above analysis: for this reason artificially constructed curves were used in determination of %MOD. At BW12C doses of 20 mg/kg, up to 23% of the haemoglobin was modified to a high-affinity form, approaching levels of theoretical clinical benefit in sickle-cell disease.3 Modified haemoglobin is unavailable for oxygen transport, but no clinical or biochemical evidence of tissue
hypoxia was seen. Despite the documented anti-sickling activity ofBW 12C in vitro,4,6 it would be surprising to see a reduction in the haemolytic rate over the short time-scale of this study. Interpretation of the biochemical changes is hampered by the lack of a control group on bed rest alone. However, the small falls in serum bilirubin and aspartate aminotransferase concentrations do indicate a reduced rate ofhaemolysis in the immediate postinfusion period. Preservation of rheologically compromised erythrocytes could explain the impairment in erythrocyte filtrability observed at 4 h; in-vitro studies have demonstrated the high sensitivity of IF to increasing percentages of irreversibly sickled cells added to sickle whole blood These observations suggest that short-term
834
infusions of BW12C should be tailed off to prevent any rebound increase in sickling caused by the temporarily longer survival of rheologically compromised cells. An analogue with a longer half-life might overcome this difficulty. Progressive deoxygenation of sickle blood in vitro hampers filtration of erythrocytes under positive pressure through 5 m diameter pores. This impairment can be prevented by the in-vitro addition of BW12C concentrations ofl’5mmol/1 and above.6 Similarly, the initial-flow-rate gravity filtration method used in the present study showed a progressive fall in the critical P02 for filtration when BW12C was added in increasing concentration (0-3 mmol/1) to sickle blood invitro. Only one of six subjects, however, showed a fall in the critical P02 at a BW12C concentration off 75 mmol/1 (the maximum whole blood concentration achieved in vivo with intravenous infusion). This result, and the apparent absence of rheological benefit in the in-vivo study, suggests that a quantitatively greater left-shift of the oxygen saturation curve may be required to secure rheological improvement in sickle cells of a degree that can be detected by our existing filtration
technique. Further studies of BW12C at higher doses for longer periods, both in the steady state and during crisis, will be ’needed to evaluate the role of this agent in the treatment of sickle-cell disease. In these investigations, patients should be carefully monitored to ensure that "therapeutic" levels, as regards sickling, do not compromise oxygen delivery to tissues. Any reduction in oxygen delivery, in conjunction with reduced haemolysis, might cause a rise in erythrocyte mass and hence whole blood viscosity; thus rheological monitoring will be required to ensure that the rise in whole blood viscosity does not overwhelm the rheological benefit of increased
erythrocyte deformability.6
This work was supported by the Clinical and Applied Research Division of the Wellcome Foundation Ltd. A. J. K. is funded by Action Research for the Crippled Child. We are indebted to Dr M. Toop, Dr R. G. Sparks, and Mr S. S. Marwah for laboratory analyses; to Dr R. Wootton for help in the analysis of oxygen saturation curves; to Dr D. Bareford, Dr G. S. Lucas, and Dr J. Porter for patient supervision in the early stages of the study; and to the eight volunteers.
Correspondence should be addressed to J. S., Department of Haematology, University of Birmingham, Birmingham B15 2TJ.
Medical School,
REFERENCES ER. Treatment of sickle-cell disease. Trans R Soc Trop Med Hyg 1974; 68: 85-91. 2. Brittenham GM, Schechter AN, Noguchi CT. Hemoglobin S polymerization: primary determinant of the hemolytic and clinical severity of the sickling syndromes. Blood 1.
May A, Huehns
1985; 65: 183-89.
IM, Rosemeyer MA, Huehns ER. Sickle cell disease: the proportion of J 1983; 54: 579-87. liganded haemoglobin needed to prevent crises. Br Haematol 4. Beddell CR, Goodford PJ, Kneen G, White RD, Wilkinson S, Wootton R. Substituted benzaldehydes designed to increase the oxygen affinity of human haemoglobin and inhibit the sickling of sickle erythrocytes. Br J Pharmacol 1984; 82: 397-407. 5. Fitzharris P, McLean AEM, Sparks RG, Weatherley BC, White RD, Wootton R. The effects in volunteers of BW12C, a compound designed to left-shift the blood-oxygen saturation curve. Br J Clin Pharmacol 1985; 19: 471-81. 6. Kenny MW, Stuart J. Preservation of deformability (filterability) of sickle cells by BW12C during progressive deoxygenation. Br J Haematol 1983; 55: 465-71. 7. Dacie JV, Lewis SM. Practical haematology, 6th ed. Edinburgh: Churchill Livingstone, 1984. 8. Bessis M. Red cell shapes. An illustrated classification and its rationale. Nouv Rev Fr 3. Franklin
Hématol 1972; 12:
The
Management of AIDS Patients
Edited by D. Miller, J. Weber, and J. Green, St Mary’s Hospital, London. London: Macmillan. 1986. Pp 195. ,E30.
IT is hard to name a disease that has had a greater impact on in general, and the teaching hospital in particular, than AIDS. Moreover, we should try to understand why certain teaching hospitals in major cities become "AIDS central" and suffer most upheaval and readjustment. Several factors determine this phenomenon-usually the physical proximity of the institution to its AIDS risk-group clients and the expertise of the existing staff in providing service for an AIDS risk-group or for any of the clinical expressions of AIDS virus infection. St Mary’s, London, is an example of a hospital where many of these factors’have operated. In this book staff from St Mary’s outline their approach to the diagnosis and treatment of persons with AIDS and related illnesses, based on their extensive experience in venereology. For the relatively well seropositive person the venereology model works because the staff are experienced and comfortable dealing non-judgmentally with outpatient clients; the chapter written outside St Mary’s, in which haemophiliacs are seen as "innocent victims" of AIDSvirus infection, reflects a more negative attitude. When, however; the client becomes immunodeficient and requires the services of the teaching hospital department of internal medicine and its subspecialties, the venereology model is unhelpful and this book shows how effectively the changing needs of the client can be integrated in one institution. Overall, I enjoyed it. The emphasis of each chapter varies appropriately in relation to its predominant audience and the medical and scientific aspects are generally comprehensive and sound. In view of the prediction that the Centers for Disease Control surveillance definition will collapse as the dreadful prognosis of severe immune deficiency becomes accepted, I believe that the authors are overzealous in requiring a proven diagnosis of opportunistic infection in order to fulfil a rigid case definition of end-stage AIDS. Good clinical illustration is an important aspect of such a practical book. Several of the colour reproductions are unfortunate; for example, the photograph of Kaposi’s sarcoma on the buttocks also showing a tattoo is a better demonstration of British patriotism than of the characteristic appearance of the tumour, and the picture of the cotrimoxazole rash in a ventilated patient negatively reinforces an aggressive approach to management. The section on nursing aspects, emphasising total patient care, is carefully prepared and pragmatic. Although I found the patient problem analysis tables repetitive and unimaginative, the chapters on the psychosocial aspects were excellent. The suggestion that prognosis or statistics should not be discussed at initial diagnosis of full-blown AIDS strikes me as impractical as well
society
as
deceitful.
Time will tell whether the St Mary’s framework for management of AIDS virus infection is a major model or only one of many that can provide comprehensive, compassionate, and cost-effective care. I recommend this book to any professional who is involved in the care of these persons. Centre for Immunology, St Vincent’s Hospital, Sydney, NSW 2010, Australia
12.
721-46.
DAVID A. COOPER
Davis LR, Huehns ER, White JM. Survey of sickle cell disease in England and Wales Br Med J 1981; 283: 1519-21. Thomas AN, Pattison C, Serjeant GR. Causes of death in sickle cell disease in Jamaica Br Med J 1982; 285: 633-35. Stuart J. Sickle cell disease and vascular occlusion-rheological aspects. Clin Hemorheol 1984; 4: 193-207. Dean J, Schechter AN. Sickle cell anemia: molecular and cellular bases of therapeutic approaches. N Engl J Med 1978; 299: 752-65, 804-11, 863-70. Harkness DR, Roth S. Clinical evaluation of cyanate in sickle cell anemia. Prog Hematol
Erythrocyte filtrability measurement by the initial flow rate method. Biorheology 1983; 20: 199-211. Stuart J, Stone PCW, Bareford D, Caldwell NM, Davies JE, Baar S. Evaluation of leucocyte removal methods for studies oferythrocyte deformability. Clin Hemorheol
13.
1985; 5: 137-47. GS, Caldwell NM, Stuart J. Fluctuating deformability of oxygenated sickle erythrocytes in the asymptomatic state and in painful crisis. Br J Haematol 1985; 59:
15.
363-68.
1975; 9: 157-84. 17. Stuart J, Keidan AJ, Marwah SS, Green MA. Rheological methods for the evaluation of anti-sickling compounds in clinical trials. In: Beuzard Y, Charache S, Galacteros F, eds. Approaches to the therapy of sickle cell disease. INSERM Symp (in press).
9. Hanss M. 10.
Reviews of Books
14.
11. Lucas
16.