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GRADIENT IN CHOLESTEROL CONCENTRATION ACROSS HUMAN AORTIC WALL
1254
hanging bepeath the bed. The suction required (by lowering the ends of the tubes) is approximately 10 cm. per 15 ml. per min. rate of flow. Slight bloodstaining of the specimens is usual; this is probably owing to suction on the ureteric mucosa as it increases with lowering of the
protein.45 Autoradiographic evidence obtained after injection of 8H-cholesterol suggests that lipids diffuse or are transported across the aortic wall of the rat and rabbit.3 This outward transport of labelled lipid was still observed after extirpation of the vasa vasorum of
tube ends. The investigation is then begun by setting up an appropriate intravenous infusion, and the renal specimens are collected for one or two hours. The bladder is
rabbit aorta. Histochemical stains on the aorta in both human atherosclerosis (fig. 1) and cholesterol-induced rabbit atheroma (fig. 2) show that lipid deposition is not confined to the tunica intima but may also occur in the tunica media. These observations led us to consider whether a gradient of lipid concentration may exist across the arterial wall and whether this gradient may be exaggerated in atherosclerosis. In this communication we present analyses of cholesterol and phospholipid in multiple layers of human aortic wall, cut at uniform thickness from the inside to the outside of the vessel.
compressed suprapubically at quarter-hourly intervals and finally at the end of the procedure, after the foot of the bed has been lowered, to confirm the absence of any leak around the tubes into the bladder. Afterwards retrograde pyelograms can be performed if the previous intravenous pyelogram has not been satisfactory. The estimations of suction required for different rates of urine flow were done by Dr. D. N. S. Kerr, who has carried out all the renal function studies.
Preliminary
Communication
Samples of human lower thoracic aorta were obtained at pinned out flat on cork, and fixed in calcium acetate (1 %)/formalin (10%). A square was cut from the flattened fixed aorta, weighed, and frozen on to the chuck of a De La Rue Frigistor thermoelectric (controlled temperature) microtome stage cooler (model FS 1). The inner part of atheromatous lesions necropsy,
trimmed with the microtome until the inner aspect of the tunica intima presented a uniformly flat surface; the block was then allowed to thaw and was again weighed. After re-freezing the block on to the chuck, sections were cut at 50(i and were arranged in groups of equal numbers representing different layers of the aortic wall. For different aortas, the numbers of sections in each layer varied from 2(100[1.) to 4(200[1.); between five and eleven layers were thus obtained from each sample of aorta. The region of the intimomedial
was
GRADIENT IN CHOLESTEROL CONCENTRATION ACROSS HUMAN AORTIC WALL IT has been suggested that lipid may be deposited in the arterial wall as a result either of failure in transport 1-3 across the wall or of instability of infiltrated lipoAdams, C. W. M., Bayliss, O. B., Ibrahim, M. Z. M. Lancet, 1962, i, 890. Adams, C. W. M. in Symposium on Biological Aspects of Occlusive Vascular Disease (edited by D. G. Chalmers and G. A. Gresham). London, 1963. Adams, C. W. M., Bayliss, O. B., Davision, A. N., Ibrahim, M. Z. M. J. Path. Bact. (in the press).
1. 2.
3.
Fig. 1-Accumulation
of
esters
in intima
mark intimomedial junction.
(Osmium
cholesterol/triglyceride
and media of atherosclerotic human aorta.
Intima
at
top;
arrows
tetroxide-fx-naphthylamine. X 40.)
4. 5.
Page, I. H. Circulation, 1954, 10, 1. Kellner, A. in Symposium on Atherosclerosis, National Academy of Sciences, National Research Council, Washington, D.C., 1954.
Fig. 2-Accumulation of cholesterol
in intima and inner media of rabbit aorta in cholesterol-induced atheroma.
Intima
at
top;
arrows
acid/naphthoquinone.
X
mark intimomedial
80.)
junction. (Perchloric-
1255 TABLE I-CHOLESTEROL IN MULTIPLE LAYERS OF HUMAN AORTIC WALL (mg. per 100 mg. wet weight)
’W.H.O. classification: >2
mm.
grade i = lesions < 2 mm. diam.; grade diam.; grade H!== complicated lesions.
m
= lesions
was recognised by inspection of the side of the block, where it appeared as a thin yellow line separating these two layers. Since the sections were cut at 50. and in the circumferential plane of the aorta, histological identification of the junction was unsatisfactory. It was established that the sections of aortic wall cut on this constant-temperature microtome were of uniform thickness; for serial sections cut from a cube of normal rat liver-an organ of nearly homogeneous histological structure-showed only 4-5% coefficient of variation in weight. The sections constituting each layer were homogenised in chloroform/methanl (2/1 v/v) and were equilibrated with water. The chloroform phase was removed, made up to volume, and divided into aliquots; these aliquots were analysed for lipid phosphorus’ and total cholesterol.’7 In experiments to be described elsewhere, it was found that preliminary fixation in formalin did not significantly reduce the amount of total phospholipid and cholesterol that could be recovered from the tissue.
junction
The results are summarised in tables I and 11. Except in cases 1, 3, and 9, the analyses for cholesterol, (table i) showed a definite gradient in concentration of cholesterol from inside to outside of the aortic wall; this gradient tended to be more prominent in vessels with more severe atherosclerosis. Estimations of phospholipid, however, did not usually reveal a gradient in concentration of this lipid from inside to outside of the aortic wall, and, even when a slight gradient was found (cases 1, 5, 10), it was of much lower order than that of cholesterol. Accordingly, the phospholipid/cholesterol ratio was found actually to rise from inside to outside of the vessel wall (table 11), except in case 5. 6. 7.
Folch, J., Lees, M., Sloane-Stanley, G. H. J. biol. Chem. 1957, 226, Davison, A. N., Wajda, M. Biochem. J. 1962, 82, 113.
TABLE
II-PHOSPHOLIPID/CHOLESTEROL
RATIO IN MULTIPLE OF HUMAN AORTIC WALL
497.
LAYERS
These results show that cholesterol tends to accumulate in the layers of the human aortic wall in inverse proportion to the distance of each layer from the lumen, and that cholesterol accumulates in the tunica media, as well as in the tunica intima, of the atherosclerotic human aorta. Our observations do not indicate whether entry of lipid into the tunica media is due to metabolic and mechanical obstruction to the normal diffusion of lipid across the aortic wall or whether it results from an abnormal infiltration of lipid into the tunica media. The absence of a transport or diffusion gradient for phospholipid across the aortic wall is consistent with the view that phospholipid is synthesised within the arterial wall 8-11 and that it is a normal component of the elastic lamellar of the aortic media. 12 This work was supported by U.S. P.H.S. grants nos. HE-06483-02 and 03 of the National Heart Institute, National Institutes of Health, Bethesda, Maryland. JAMES N. DAVIS B.A.
New York
Research Student
C. W. M. ADAMS M.A., M.D., Cantab
Department of Pathology, Guy’s Hospital Medical School, London, S.E.1, and Cornell University Medical College,
Reader in
Experimental Pathology
O. B. BAYLISS A.I.M.L.T.
Research Technician
New York
Hypothesis HYPOTHERMIA, LYMPHOPENIA,
AND THE
IMMUNOLOGICAL STATE OBSERVATIONS
during the past three years on patients undergoing prolonged mild whole-body hypothermia (30-32°C) indicate that the response of the different white cells to cooling varies. The number of circulating neutrophils may rise, fall, or remain relatively unaltered, but the lymphocytes and monocytes show a profound fall, and the eosinophils and basophils may disappear completely from the circulating blood within 24-48 hours. These changes are present throughout the period of hypothermia and often persist during the first 2-4 days of rewarming. The peripheral blood-picture therefore is of a lymphopenia, with a variable neutrophil-count. It may be thought that the lymphopenia results from an increase in adrenocortical activity, but both clinical and experimental evidence are against this. Moreover, the lymphopenia associated with increased adrenocortical activity, unlike that of hypothermia, is of limited duration,-3 the lymphocytes returning to normal within 24 hours despite a sustained increase in secretion of adrenal cortical hormone.14 Urinary 17-hydroxycorticosteroid levels show considerable fluctuation during hypothermia but are mostly lower than at normal temperature. This observation is in keeping with that of Hume and Bell," who reported a reduction in the minuteoutput of 17-hydroxycorticosteroids in the adrenal venous blood of a patient during surgery at 30°C, and of Hume and Egdahl," who reported similar findings in dogs, commencing at temperatures below 34-35°C, and 10. 11. 12. 13.
Zilversmit, D. B., McCandless, E. L. J. lipid Res. 1959, 1, 118. Zilversmit, D. B., McCandless, E. L., Jordan, P. H., Henly, W. S., Ackerman, R. H. Circulation, 1961, 23, 370. Christensen, S. J. J. Atheroscler. Res. 1962, 2, 131. Adams, C. W. M., Tuqan, N. A. J. Path. Bact. 1961, 82, 131. Adams, C. W. M., Bayliss, O. B. ibid. 1963, 85, 113. Britton, C. J. C. in Whitby and Britton’s Disorders of the Blood; p. 78.
14. 15. 16.
London, 1963. Hills, A. G., Forsham, P. H., Finch, C. A. Blood, 1948, 3, 755. Hume, D. M., Bell, C. C. Surg. Forum, 1958, 9, 6. Hume, D. M., Egdahl, R. H. Ann. N. Y. Acad. Sci. 1959, 80, 435.
8. 9.
hanging bepeath the bed. The suction required (by lowering the ends of the tubes) is approximately 10 cm. per 15 ml. per min. rate of flow. Slight bloodstaining of the specimens is usual; this is probably owing to suction on the ureteric mucosa as it increases with lowering of the
protein.45 Autoradiographic evidence obtained after injection of 8H-cholesterol suggests that lipids diffuse or are transported across the aortic wall of the rat and rabbit.3 This outward transport of labelled lipid was still observed after extirpation of the vasa vasorum of
tube ends. The investigation is then begun by setting up an appropriate intravenous infusion, and the renal specimens are collected for one or two hours. The bladder is
rabbit aorta. Histochemical stains on the aorta in both human atherosclerosis (fig. 1) and cholesterol-induced rabbit atheroma (fig. 2) show that lipid deposition is not confined to the tunica intima but may also occur in the tunica media. These observations led us to consider whether a gradient of lipid concentration may exist across the arterial wall and whether this gradient may be exaggerated in atherosclerosis. In this communication we present analyses of cholesterol and phospholipid in multiple layers of human aortic wall, cut at uniform thickness from the inside to the outside of the vessel.
compressed suprapubically at quarter-hourly intervals and finally at the end of the procedure, after the foot of the bed has been lowered, to confirm the absence of any leak around the tubes into the bladder. Afterwards retrograde pyelograms can be performed if the previous intravenous pyelogram has not been satisfactory. The estimations of suction required for different rates of urine flow were done by Dr. D. N. S. Kerr, who has carried out all the renal function studies.
Preliminary
Communication
Samples of human lower thoracic aorta were obtained at pinned out flat on cork, and fixed in calcium acetate (1 %)/formalin (10%). A square was cut from the flattened fixed aorta, weighed, and frozen on to the chuck of a De La Rue Frigistor thermoelectric (controlled temperature) microtome stage cooler (model FS 1). The inner part of atheromatous lesions necropsy,
trimmed with the microtome until the inner aspect of the tunica intima presented a uniformly flat surface; the block was then allowed to thaw and was again weighed. After re-freezing the block on to the chuck, sections were cut at 50(i and were arranged in groups of equal numbers representing different layers of the aortic wall. For different aortas, the numbers of sections in each layer varied from 2(100[1.) to 4(200[1.); between five and eleven layers were thus obtained from each sample of aorta. The region of the intimomedial
was
GRADIENT IN CHOLESTEROL CONCENTRATION ACROSS HUMAN AORTIC WALL IT has been suggested that lipid may be deposited in the arterial wall as a result either of failure in transport 1-3 across the wall or of instability of infiltrated lipoAdams, C. W. M., Bayliss, O. B., Ibrahim, M. Z. M. Lancet, 1962, i, 890. Adams, C. W. M. in Symposium on Biological Aspects of Occlusive Vascular Disease (edited by D. G. Chalmers and G. A. Gresham). London, 1963. Adams, C. W. M., Bayliss, O. B., Davision, A. N., Ibrahim, M. Z. M. J. Path. Bact. (in the press).
1. 2.
3.
Fig. 1-Accumulation
of
esters
in intima
mark intimomedial junction.
(Osmium
cholesterol/triglyceride
and media of atherosclerotic human aorta.
Intima
at
top;
arrows
tetroxide-fx-naphthylamine. X 40.)
4. 5.
Page, I. H. Circulation, 1954, 10, 1. Kellner, A. in Symposium on Atherosclerosis, National Academy of Sciences, National Research Council, Washington, D.C., 1954.
Fig. 2-Accumulation of cholesterol
in intima and inner media of rabbit aorta in cholesterol-induced atheroma.
Intima
at
top;
arrows
acid/naphthoquinone.
X
mark intimomedial
80.)
junction. (Perchloric-
1255 TABLE I-CHOLESTEROL IN MULTIPLE LAYERS OF HUMAN AORTIC WALL (mg. per 100 mg. wet weight)
’W.H.O. classification: >2
mm.
grade i = lesions < 2 mm. diam.; grade diam.; grade H!== complicated lesions.
m
= lesions
was recognised by inspection of the side of the block, where it appeared as a thin yellow line separating these two layers. Since the sections were cut at 50. and in the circumferential plane of the aorta, histological identification of the junction was unsatisfactory. It was established that the sections of aortic wall cut on this constant-temperature microtome were of uniform thickness; for serial sections cut from a cube of normal rat liver-an organ of nearly homogeneous histological structure-showed only 4-5% coefficient of variation in weight. The sections constituting each layer were homogenised in chloroform/methanl (2/1 v/v) and were equilibrated with water. The chloroform phase was removed, made up to volume, and divided into aliquots; these aliquots were analysed for lipid phosphorus’ and total cholesterol.’7 In experiments to be described elsewhere, it was found that preliminary fixation in formalin did not significantly reduce the amount of total phospholipid and cholesterol that could be recovered from the tissue.
junction
The results are summarised in tables I and 11. Except in cases 1, 3, and 9, the analyses for cholesterol, (table i) showed a definite gradient in concentration of cholesterol from inside to outside of the aortic wall; this gradient tended to be more prominent in vessels with more severe atherosclerosis. Estimations of phospholipid, however, did not usually reveal a gradient in concentration of this lipid from inside to outside of the aortic wall, and, even when a slight gradient was found (cases 1, 5, 10), it was of much lower order than that of cholesterol. Accordingly, the phospholipid/cholesterol ratio was found actually to rise from inside to outside of the vessel wall (table 11), except in case 5. 6. 7.
Folch, J., Lees, M., Sloane-Stanley, G. H. J. biol. Chem. 1957, 226, Davison, A. N., Wajda, M. Biochem. J. 1962, 82, 113.
TABLE
II-PHOSPHOLIPID/CHOLESTEROL
RATIO IN MULTIPLE OF HUMAN AORTIC WALL
497.
LAYERS
These results show that cholesterol tends to accumulate in the layers of the human aortic wall in inverse proportion to the distance of each layer from the lumen, and that cholesterol accumulates in the tunica media, as well as in the tunica intima, of the atherosclerotic human aorta. Our observations do not indicate whether entry of lipid into the tunica media is due to metabolic and mechanical obstruction to the normal diffusion of lipid across the aortic wall or whether it results from an abnormal infiltration of lipid into the tunica media. The absence of a transport or diffusion gradient for phospholipid across the aortic wall is consistent with the view that phospholipid is synthesised within the arterial wall 8-11 and that it is a normal component of the elastic lamellar of the aortic media. 12 This work was supported by U.S. P.H.S. grants nos. HE-06483-02 and 03 of the National Heart Institute, National Institutes of Health, Bethesda, Maryland. JAMES N. DAVIS B.A.
New York
Research Student
C. W. M. ADAMS M.A., M.D., Cantab
Department of Pathology, Guy’s Hospital Medical School, London, S.E.1, and Cornell University Medical College,
Reader in
Experimental Pathology
O. B. BAYLISS A.I.M.L.T.
Research Technician
New York
Hypothesis HYPOTHERMIA, LYMPHOPENIA,
AND THE
IMMUNOLOGICAL STATE OBSERVATIONS
during the past three years on patients undergoing prolonged mild whole-body hypothermia (30-32°C) indicate that the response of the different white cells to cooling varies. The number of circulating neutrophils may rise, fall, or remain relatively unaltered, but the lymphocytes and monocytes show a profound fall, and the eosinophils and basophils may disappear completely from the circulating blood within 24-48 hours. These changes are present throughout the period of hypothermia and often persist during the first 2-4 days of rewarming. The peripheral blood-picture therefore is of a lymphopenia, with a variable neutrophil-count. It may be thought that the lymphopenia results from an increase in adrenocortical activity, but both clinical and experimental evidence are against this. Moreover, the lymphopenia associated with increased adrenocortical activity, unlike that of hypothermia, is of limited duration,-3 the lymphocytes returning to normal within 24 hours despite a sustained increase in secretion of adrenal cortical hormone.14 Urinary 17-hydroxycorticosteroid levels show considerable fluctuation during hypothermia but are mostly lower than at normal temperature. This observation is in keeping with that of Hume and Bell," who reported a reduction in the minuteoutput of 17-hydroxycorticosteroids in the adrenal venous blood of a patient during surgery at 30°C, and of Hume and Egdahl," who reported similar findings in dogs, commencing at temperatures below 34-35°C, and 10. 11. 12. 13.
Zilversmit, D. B., McCandless, E. L. J. lipid Res. 1959, 1, 118. Zilversmit, D. B., McCandless, E. L., Jordan, P. H., Henly, W. S., Ackerman, R. H. Circulation, 1961, 23, 370. Christensen, S. J. J. Atheroscler. Res. 1962, 2, 131. Adams, C. W. M., Tuqan, N. A. J. Path. Bact. 1961, 82, 131. Adams, C. W. M., Bayliss, O. B. ibid. 1963, 85, 113. Britton, C. J. C. in Whitby and Britton’s Disorders of the Blood; p. 78.
14. 15. 16.
London, 1963. Hills, A. G., Forsham, P. H., Finch, C. A. Blood, 1948, 3, 755. Hume, D. M., Bell, C. C. Surg. Forum, 1958, 9, 6. Hume, D. M., Egdahl, R. H. Ann. N. Y. Acad. Sci. 1959, 80, 435.
8. 9.