- Email: [email protected]
IRON POISONING: ANOTHER ENERGY CRISIS
664 drome opens a new chapter in our understanding of the role played by that essential trace element in the body generally and in maintaining the integrity of skin in parti-
proliferation (even to the point of neoplastic transformationj would not be unexpected.lo In addition to cytopathic changes from the virus itself,
cular.
one
would expect a cell-mediated immune response directed against the virus. Later a humoral (antibody) response might give rise to an elevation in the immunoglobulin G level in brain and cerebrospinal fluid. An autoimmune response could be induced, but it seems more likely that continued viral presence would episodically trigger the whole process of direct virus-induced cytolysis with secondary immune response. Moreover, lymphocytes elaborating lymphokines or antibodies which destroy a virus might inadvertently cause tissue destruction as well. If there is an analogy between M.s. and herpesvirus infections we should be seeking virus from the eye, the trigeminal, geniculate, and dorsal-root ganglia. We can expect to find herpesviruses-and they may be important in the aetiology and pathogenesis of multiple sclerosis. But we would find herpesvirus in many normal ganglia.ll Will we find some other viruses in M.s. specimens ?
BENJAMIN PORTNOY
Skin Hospital, Manchester 3.
MOHAMED MOLOKHIA.
MULTIPLE SCLEROSIS: A FEVER BLISTER ON THE BRAIN? SIR,—There are certain similarities between infection by Herpes labialis (Herpesvirus hominis type 1, H.V.H.) and multiple (disseminated) sclerosis (M.s.). Both are characterised by recrudescences or relapses, which tend to recur in the same anatomical region. Attacks in both diseases seem at times to be precipitated by emotional stress, infections, exposure to irradiation, fever, or trauma.l.1I Perhaps in addition to clinical similarities there are similarities in pathological physiology. H.v.H. enters the nervous system primarily by intraaxonal passage. 3-6 In addition to, or instead of, causing severe encephalitis, the virus may enter a latent state and remain undetectable in neurons.6 A recrudescence of labial or ophthalmic lesions occurs when the virus moves centrifugally in the axon and enters permissive epithelial cells.’ The virus is now detectable as a complete infectious virion and cytolysis occurs. There is meagre evidence for neuronal involvement in
Department of Neurology, Reed Neurological Research Center, UCLA School of Medicine, Los Angeles, California 90024, U.S.A.
IRON POISONING: ANOTHER ENERGY CRISIS
multiple sclerosis-rarely, a seizure, trigeminal neuralgia, a radiculopathy ", anti-neuronal antibodies.8 However,. if the myelin-supporting oligodendroglia cell were permissive or partially permissive for any virus (not necessarily "
e
herpesvirus) then
the neuron might serve as the reservoir of latent virus, the genome of which could pass to the oligodendrocyte. The neuron will not exhibit many changes, but the oligodendroglia cell (perhaps analogous to the epithelial cell in H.v.H.) could. If complete virions were formed one might see cytolysis and intact virions
electron
microscopically. However, if abortive infection occurred, membrane changes in antigenicity might be the only detectable abnormality of the glia cells. If the virus were activated and moved centripetally in the axon rather than centrifugally it would encounter permissive cells and become cytopathic. For example, centripetal passage from retinal neurons would give rise to retrobulbar neuritis; from the trigeminal ganglion would give rise to trigeminal-tract destruction; from a dorsal-root ganglion would give rise to a myelopathy. More central projections of peripheral neurons would serve as loci for subcortical white matter demyelination. A similar phenomenon can occur in Herpesvirus varicellazoster.9
Persisting virus would cause other changes. If the virus eventually interfered with vital cell processes, direct neuronal damage would occur and might explain the chronic progressive state seen in M.s. of long duration. By mechanisms not understood, virus-induced glial M., Lumsden, C. E., Acheson, E. D. Multiple Reappraisal. Baltimore, 1972. 2. Juel-Jensen, B. E., Maccallum, F. D. Herpes Simplex Varicella and Zoster. Philadelphia, 1972. 3. Goodpasture, E. W., Teague, O. J. med. Res. 1923, 44, 139. 4. Knotts, F. B., Cook, M. L., Stevens, J. G. J. exp. Med. 1973, 138, 740. 5. Knotts, F. B., Cook, M. L., Stevens, J. G. J. infect. Dis. (in the 1.
McAlpine,
Sclerosis:
D. a
press). Paine, T. F., Jr. Bact. Rev. 1964, 28, 472. 7. Carton, C. A. J. Neurosurg. 1953, 10, 463. 8. Bomstein, M. B. in Multiple Sclerosis (edited by F. Wolfgram, G. W. Ellison, J. H. Stevens, and J. M. Andrews); p. 123. New York, 1972. 9. Hogan, E. L., Krigman, M. R. Archs Neurol. 1973, 29, 309. 6.
GEORGE W. ELLISON.
SIR,-A therapeutic boon for one patient may lead to tragic consequences for another. Iron tablets, legitimately prescribed for a mother and packaged in non-childproof bottles, often appear similar to the green and brown candies known as M & M’s and are largely responsible for the approximately 2000 accidental iron poisonings in the United States annually. The vast majority of these poisonings occur in children, and, the morbidity in published series approaches 85%. Symptoms of iron intoxication include vomiting, haematemesis, diarrhoea, lethargy, coma, irritability, seizures, and abdominal pain. Signs "
"
may include
an increased cardiac and respiratory rate, while a marked increase in total peripheral vascular resistance may maintain arterial blood-pressure for variable periods before manifestations of low-output shock ensue. Biochemical consequences of acute iron intoxication include metabolic acidosis, due in part to the accumulation of lactic and citric acids, and hypoglycxmia. Since the pathogenesis of iron poisoning has not been established,’ we wish to offer some speculative thoughts in the hope of stimulating interest in the problem. Ganote and Nahara 12 have shown that rats given oral toxic doses of ferrous sulphate develop lethargy, diarrhoea, and peripheral vasoconstriction with only a mild decrease in blood-pressure. Electron microscopy of the liver reveals that the mitochondrion is the initial and primary site of intracellular injury. The first detectable morphological change in hepatocytes is the appearance of electron-dense deposits, presumably iron, between the inner and outer mitochondrial membranes as well as in the intracristal lumen and the matrix. These deposits are visible before other evidence of cellular damage. Although the mitochondrial membrane is not normally freely permeable to the ferric ion, the massive quantities present in iron poisoning may facilitate entry into the matrix due to the increased concentration gradient or possibly to alterations in membrane permeability as a result of iron-mediated
lipid peroxidation. These morphological changes can be correlated with in-vitro assays demonstrating impaired cellular function 10. Reagan, T. J., Freiman, I. S. J. Neurol. Neurosurg. Psychiat. 1973, 36, 523. 11. Baringer, J. R., Swoveland, P. New Engl. J. Med. 1973, 288, 648. 12. Ganote, C. E., Nahara, G. Lab. Invest. 1973, 28, 426.
665
Suizzested ferric-reductase shunt.
the mitochondrial level. Normally, the addition of a substrate for the Krebs cycle to an intact mitochondrial preparation leads to a very slow rate of oxygen uptake, reflecting the minimal movement of electrons down the electron-transport chain. When adenosine diphosphate is added in addition to the Krebs-cycle substrate, oxygen uptake is very rapid. These mitochondria are said to exhibit respiratory control. As early as three hours after the ingestion of a toxic quantity of iron, Ganote and Nahara were able to demonstrate an increased rate of oxygen uptake in the presence of a Krebs-cycle substrate alone, and a decreased rate of oxygen uptake after the additional provision of adenosine diphosphate. The rates of oxygen uptake in both these situations were nearly the same in iron-poisoned animals. Thus, in iron-poisoned mitochondria, the addition of a substrate produces electron transport and oxygen uptake, while the additional provision of adenosine diphosphate does not lead to a further increase in oxygen uptake. These poisoned mitochondria can be said to lack respiratory control. The basis of these in-vitro perturbations in mitochondrial function might be explained, at least in part, by a report of Barnes et al. on mitochondrial iron metabolism.l3 These workers described a previously unknown enzymatic activity, which we shall refer to as ferric reductase, associated with the inner surface of the inner mitochondrial membrane and catalysing the reduction of ferric iron to ferrous iron. Iron in the ferrous state is the substrate for ferrochelatase, which inserts iron into protoporphyrin ix to form heme. It seems reasonable that, in the presence of excess iron, the ferric reductase could serve as a shunt for electrons, removing them from their usual pathway and donating them to the ferric iron. The ferrous iron produced might then be oxidised by oxygen, setting up a cycle as illustrated in the accompanying figure. Such a shunt is suggested by the experimentally demonstrated oxygen uptake by mitochondria provided with ferric ions in the presence of rotenone, which blocks electron flow between a flavoprotein and co-enzyme Q. Oxygen is then taken up when the ferrous iron generated via the ferric-reductase shunt is auto-oxidised back to ferric iron within the mitochondrial matrix. Although not initially recognised, ferric reductase may thus have pivotal importance in the clinical, morphological, and biochemical consequences of iron intoxication. Given unlimited supplies of the ferric ion, the enzyme could preferentially catalyse the shunting of electrons away from the electron-transport chain. This would be feasible since the reduction of ferric iron is thermodynamically favoured over the channelling of electrons through the conventional cytochrome system. This surmise is consistent with the demonstrated loss of respiratory control in liver mitochondria from iron-poisoned animals and would explain at
13.
Barnes, R., Connelly, J. L., Jones, O. T. G. Biochem. J. 1972, 128, 1043.
the changes observed in the rates of oxygen uptake in the presence and absence of adenosine diphosphate. An active ferric-reductase shunt would result directly in immediate cessation of aerobic adenosine-triphosphate synthesis, precipitating a cellular energy crisis. This could be correlated with the elevated lactic and citric acid levels and the depletion of glycogen stores observed in iron poisoning. Ultimately, failure to synthesise sufficient adenosine triphosphate could lead to cell death. Obviously the ferric-reductase shunt need not be localised exclusively to liver mitochondria, and a similar explanation might be applicable to the cardiovascular, neurological, and gastrointestinal manifestations associated with iron intoxication. The existence of the ferric-reductase shunt may provide a point of departure for further investigation of the biochemical mechanism of acute iron toxicity and may have a
bearing
on
treatment.
Boston University School of
Medicine, City Hospital, Boston, Massachusetts, U.S.A. Boston University School of Boston
JAMES L. ROBOTHAM. ROBERT F. TROXLER.
Medicine.
Johns Hopkins University School Medicine, Baltimore, Maryland.
o
PAUL S. LIETMAN.
ST-SEGMENT DISPLACEMENT AFTER ACUTE MYOCARDIAL INFARCTION
SIR,-The article by Dr Morris and his colleagues (Aug. 17, p. 372) requires comment. They state that STsegment displacement at 48 hours provides no additional information for predicting those patients in whom complications subsequently develop over a six-week period. This statement may be misconstrued. Certainly no statistician will believe that Morris et al. have proved the absence of a relationship between the ST-segment shift and the development of late ventricular dysrhythmias. On the other hand, our data clearly indicate the presence of such a relationship. In the past seven years ST-segment displacement has been used in the assessment of the duration of monitoring in the Belfast coronary-care units. 1-3 In a detailed prospective study, 187 of 466 patients were 48 hours free from risk factors evident on clinical examination.4 31 of the 187 had ST-segment displacement greater than 2 mm., and among these the incidence of late ventricular dysrhythmias was 23%. The ST segment had fallen below 2 mm. in 156 of the 187, and among these the incidence of late ventricular dysrhythmias was 1% (p < 0-001). Morris et al. do not report the incidence of late ventricular dysrhythmias among patients whose ST segment had fallen below 2 mm. at the end of 48 hours. It is, therefore, difficult to understand how they conclude that ST-segment displacement would not provide any information additional to simple clinical examination. The conclusions of Morris et al. are at variance not only with those reached in Belfast but also with the conclusions of workers in Denmark and at Harvard. Nielsen,5 in a study of 404 patients, found that the magnitude of STsegment elevation gave valuable prognostic information useful in the selection of patients for prolonged monitoring. Madias et al.6 also found that the magnitude of the ST shift correlated with the clinical course and prognosis.
at
1. 2.
3. 4. 5. 6.
Pantridge, J. F. Unpublished. Boyle, D. McC., Barber, J. M., Walsh, M. J., Shivalingappa, G., Chaturvedi, N. C. Lancet, 1972, ii, 57. Chaturvedi, N. C., Walsh, M. J., Evans, A., Munro, P., Boyle, D. McC., Barber, J. M. Br. Heart J. 1974, 36, 533. Wilson, C., Pantridge, J. F. Lancet, 1973, ii, 1284. Nielsen, B. L. Circulation, 1973, 48, 338. Madias, J. E., Venkataraman, K., Vokonas, P. S., Hood, W. B., Jr. ibid. 1973, suppl. IV, p. 195.
proliferation (even to the point of neoplastic transformationj would not be unexpected.lo In addition to cytopathic changes from the virus itself,
cular.
one
would expect a cell-mediated immune response directed against the virus. Later a humoral (antibody) response might give rise to an elevation in the immunoglobulin G level in brain and cerebrospinal fluid. An autoimmune response could be induced, but it seems more likely that continued viral presence would episodically trigger the whole process of direct virus-induced cytolysis with secondary immune response. Moreover, lymphocytes elaborating lymphokines or antibodies which destroy a virus might inadvertently cause tissue destruction as well. If there is an analogy between M.s. and herpesvirus infections we should be seeking virus from the eye, the trigeminal, geniculate, and dorsal-root ganglia. We can expect to find herpesviruses-and they may be important in the aetiology and pathogenesis of multiple sclerosis. But we would find herpesvirus in many normal ganglia.ll Will we find some other viruses in M.s. specimens ?
BENJAMIN PORTNOY
Skin Hospital, Manchester 3.
MOHAMED MOLOKHIA.
MULTIPLE SCLEROSIS: A FEVER BLISTER ON THE BRAIN? SIR,—There are certain similarities between infection by Herpes labialis (Herpesvirus hominis type 1, H.V.H.) and multiple (disseminated) sclerosis (M.s.). Both are characterised by recrudescences or relapses, which tend to recur in the same anatomical region. Attacks in both diseases seem at times to be precipitated by emotional stress, infections, exposure to irradiation, fever, or trauma.l.1I Perhaps in addition to clinical similarities there are similarities in pathological physiology. H.v.H. enters the nervous system primarily by intraaxonal passage. 3-6 In addition to, or instead of, causing severe encephalitis, the virus may enter a latent state and remain undetectable in neurons.6 A recrudescence of labial or ophthalmic lesions occurs when the virus moves centrifugally in the axon and enters permissive epithelial cells.’ The virus is now detectable as a complete infectious virion and cytolysis occurs. There is meagre evidence for neuronal involvement in
Department of Neurology, Reed Neurological Research Center, UCLA School of Medicine, Los Angeles, California 90024, U.S.A.
IRON POISONING: ANOTHER ENERGY CRISIS
multiple sclerosis-rarely, a seizure, trigeminal neuralgia, a radiculopathy ", anti-neuronal antibodies.8 However,. if the myelin-supporting oligodendroglia cell were permissive or partially permissive for any virus (not necessarily "
e
herpesvirus) then
the neuron might serve as the reservoir of latent virus, the genome of which could pass to the oligodendrocyte. The neuron will not exhibit many changes, but the oligodendroglia cell (perhaps analogous to the epithelial cell in H.v.H.) could. If complete virions were formed one might see cytolysis and intact virions
electron
microscopically. However, if abortive infection occurred, membrane changes in antigenicity might be the only detectable abnormality of the glia cells. If the virus were activated and moved centripetally in the axon rather than centrifugally it would encounter permissive cells and become cytopathic. For example, centripetal passage from retinal neurons would give rise to retrobulbar neuritis; from the trigeminal ganglion would give rise to trigeminal-tract destruction; from a dorsal-root ganglion would give rise to a myelopathy. More central projections of peripheral neurons would serve as loci for subcortical white matter demyelination. A similar phenomenon can occur in Herpesvirus varicellazoster.9
Persisting virus would cause other changes. If the virus eventually interfered with vital cell processes, direct neuronal damage would occur and might explain the chronic progressive state seen in M.s. of long duration. By mechanisms not understood, virus-induced glial M., Lumsden, C. E., Acheson, E. D. Multiple Reappraisal. Baltimore, 1972. 2. Juel-Jensen, B. E., Maccallum, F. D. Herpes Simplex Varicella and Zoster. Philadelphia, 1972. 3. Goodpasture, E. W., Teague, O. J. med. Res. 1923, 44, 139. 4. Knotts, F. B., Cook, M. L., Stevens, J. G. J. exp. Med. 1973, 138, 740. 5. Knotts, F. B., Cook, M. L., Stevens, J. G. J. infect. Dis. (in the 1.
McAlpine,
Sclerosis:
D. a
press). Paine, T. F., Jr. Bact. Rev. 1964, 28, 472. 7. Carton, C. A. J. Neurosurg. 1953, 10, 463. 8. Bomstein, M. B. in Multiple Sclerosis (edited by F. Wolfgram, G. W. Ellison, J. H. Stevens, and J. M. Andrews); p. 123. New York, 1972. 9. Hogan, E. L., Krigman, M. R. Archs Neurol. 1973, 29, 309. 6.
GEORGE W. ELLISON.
SIR,-A therapeutic boon for one patient may lead to tragic consequences for another. Iron tablets, legitimately prescribed for a mother and packaged in non-childproof bottles, often appear similar to the green and brown candies known as M & M’s and are largely responsible for the approximately 2000 accidental iron poisonings in the United States annually. The vast majority of these poisonings occur in children, and, the morbidity in published series approaches 85%. Symptoms of iron intoxication include vomiting, haematemesis, diarrhoea, lethargy, coma, irritability, seizures, and abdominal pain. Signs "
"
may include
an increased cardiac and respiratory rate, while a marked increase in total peripheral vascular resistance may maintain arterial blood-pressure for variable periods before manifestations of low-output shock ensue. Biochemical consequences of acute iron intoxication include metabolic acidosis, due in part to the accumulation of lactic and citric acids, and hypoglycxmia. Since the pathogenesis of iron poisoning has not been established,’ we wish to offer some speculative thoughts in the hope of stimulating interest in the problem. Ganote and Nahara 12 have shown that rats given oral toxic doses of ferrous sulphate develop lethargy, diarrhoea, and peripheral vasoconstriction with only a mild decrease in blood-pressure. Electron microscopy of the liver reveals that the mitochondrion is the initial and primary site of intracellular injury. The first detectable morphological change in hepatocytes is the appearance of electron-dense deposits, presumably iron, between the inner and outer mitochondrial membranes as well as in the intracristal lumen and the matrix. These deposits are visible before other evidence of cellular damage. Although the mitochondrial membrane is not normally freely permeable to the ferric ion, the massive quantities present in iron poisoning may facilitate entry into the matrix due to the increased concentration gradient or possibly to alterations in membrane permeability as a result of iron-mediated
lipid peroxidation. These morphological changes can be correlated with in-vitro assays demonstrating impaired cellular function 10. Reagan, T. J., Freiman, I. S. J. Neurol. Neurosurg. Psychiat. 1973, 36, 523. 11. Baringer, J. R., Swoveland, P. New Engl. J. Med. 1973, 288, 648. 12. Ganote, C. E., Nahara, G. Lab. Invest. 1973, 28, 426.
665
Suizzested ferric-reductase shunt.
the mitochondrial level. Normally, the addition of a substrate for the Krebs cycle to an intact mitochondrial preparation leads to a very slow rate of oxygen uptake, reflecting the minimal movement of electrons down the electron-transport chain. When adenosine diphosphate is added in addition to the Krebs-cycle substrate, oxygen uptake is very rapid. These mitochondria are said to exhibit respiratory control. As early as three hours after the ingestion of a toxic quantity of iron, Ganote and Nahara were able to demonstrate an increased rate of oxygen uptake in the presence of a Krebs-cycle substrate alone, and a decreased rate of oxygen uptake after the additional provision of adenosine diphosphate. The rates of oxygen uptake in both these situations were nearly the same in iron-poisoned animals. Thus, in iron-poisoned mitochondria, the addition of a substrate produces electron transport and oxygen uptake, while the additional provision of adenosine diphosphate does not lead to a further increase in oxygen uptake. These poisoned mitochondria can be said to lack respiratory control. The basis of these in-vitro perturbations in mitochondrial function might be explained, at least in part, by a report of Barnes et al. on mitochondrial iron metabolism.l3 These workers described a previously unknown enzymatic activity, which we shall refer to as ferric reductase, associated with the inner surface of the inner mitochondrial membrane and catalysing the reduction of ferric iron to ferrous iron. Iron in the ferrous state is the substrate for ferrochelatase, which inserts iron into protoporphyrin ix to form heme. It seems reasonable that, in the presence of excess iron, the ferric reductase could serve as a shunt for electrons, removing them from their usual pathway and donating them to the ferric iron. The ferrous iron produced might then be oxidised by oxygen, setting up a cycle as illustrated in the accompanying figure. Such a shunt is suggested by the experimentally demonstrated oxygen uptake by mitochondria provided with ferric ions in the presence of rotenone, which blocks electron flow between a flavoprotein and co-enzyme Q. Oxygen is then taken up when the ferrous iron generated via the ferric-reductase shunt is auto-oxidised back to ferric iron within the mitochondrial matrix. Although not initially recognised, ferric reductase may thus have pivotal importance in the clinical, morphological, and biochemical consequences of iron intoxication. Given unlimited supplies of the ferric ion, the enzyme could preferentially catalyse the shunting of electrons away from the electron-transport chain. This would be feasible since the reduction of ferric iron is thermodynamically favoured over the channelling of electrons through the conventional cytochrome system. This surmise is consistent with the demonstrated loss of respiratory control in liver mitochondria from iron-poisoned animals and would explain at
13.
Barnes, R., Connelly, J. L., Jones, O. T. G. Biochem. J. 1972, 128, 1043.
the changes observed in the rates of oxygen uptake in the presence and absence of adenosine diphosphate. An active ferric-reductase shunt would result directly in immediate cessation of aerobic adenosine-triphosphate synthesis, precipitating a cellular energy crisis. This could be correlated with the elevated lactic and citric acid levels and the depletion of glycogen stores observed in iron poisoning. Ultimately, failure to synthesise sufficient adenosine triphosphate could lead to cell death. Obviously the ferric-reductase shunt need not be localised exclusively to liver mitochondria, and a similar explanation might be applicable to the cardiovascular, neurological, and gastrointestinal manifestations associated with iron intoxication. The existence of the ferric-reductase shunt may provide a point of departure for further investigation of the biochemical mechanism of acute iron toxicity and may have a
bearing
on
treatment.
Boston University School of
Medicine, City Hospital, Boston, Massachusetts, U.S.A. Boston University School of Boston
JAMES L. ROBOTHAM. ROBERT F. TROXLER.
Medicine.
Johns Hopkins University School Medicine, Baltimore, Maryland.
o
PAUL S. LIETMAN.
ST-SEGMENT DISPLACEMENT AFTER ACUTE MYOCARDIAL INFARCTION
SIR,-The article by Dr Morris and his colleagues (Aug. 17, p. 372) requires comment. They state that STsegment displacement at 48 hours provides no additional information for predicting those patients in whom complications subsequently develop over a six-week period. This statement may be misconstrued. Certainly no statistician will believe that Morris et al. have proved the absence of a relationship between the ST-segment shift and the development of late ventricular dysrhythmias. On the other hand, our data clearly indicate the presence of such a relationship. In the past seven years ST-segment displacement has been used in the assessment of the duration of monitoring in the Belfast coronary-care units. 1-3 In a detailed prospective study, 187 of 466 patients were 48 hours free from risk factors evident on clinical examination.4 31 of the 187 had ST-segment displacement greater than 2 mm., and among these the incidence of late ventricular dysrhythmias was 23%. The ST segment had fallen below 2 mm. in 156 of the 187, and among these the incidence of late ventricular dysrhythmias was 1% (p < 0-001). Morris et al. do not report the incidence of late ventricular dysrhythmias among patients whose ST segment had fallen below 2 mm. at the end of 48 hours. It is, therefore, difficult to understand how they conclude that ST-segment displacement would not provide any information additional to simple clinical examination. The conclusions of Morris et al. are at variance not only with those reached in Belfast but also with the conclusions of workers in Denmark and at Harvard. Nielsen,5 in a study of 404 patients, found that the magnitude of STsegment elevation gave valuable prognostic information useful in the selection of patients for prolonged monitoring. Madias et al.6 also found that the magnitude of the ST shift correlated with the clinical course and prognosis.
at
1. 2.
3. 4. 5. 6.
Pantridge, J. F. Unpublished. Boyle, D. McC., Barber, J. M., Walsh, M. J., Shivalingappa, G., Chaturvedi, N. C. Lancet, 1972, ii, 57. Chaturvedi, N. C., Walsh, M. J., Evans, A., Munro, P., Boyle, D. McC., Barber, J. M. Br. Heart J. 1974, 36, 533. Wilson, C., Pantridge, J. F. Lancet, 1973, ii, 1284. Nielsen, B. L. Circulation, 1973, 48, 338. Madias, J. E., Venkataraman, K., Vokonas, P. S., Hood, W. B., Jr. ibid. 1973, suppl. IV, p. 195.