313
evident reasons for caution, until more facts have been gathered and assessed. Among the unanswered questions is why the reported total of children born alive with spina bifida in England and Wales has fallen from 1138 in 1970 to 678 in 19764-without routine screening. Particularly needed are better figures by which to compare the number of expected live spinabifida births with the number of normal fetuses lost either by accidental abortion after amniocentesis or by termination induced because of deceptive a-fetoprotein levels. But there
are
reliable.4 The distinct differences in net charge at a given pH result in good resolution of mixtures of C.K.
isoenzymes by zone electrophoresis or ion-exchange chromatography. Both these methods are widely used. C.K. isoenzymes, or more precisely their constituent M and B subunits, are antigenically distinct, and various immunochemical procedures have been applied to the study of C.K. isoenzymes in serum. Although all these methods are capable of giving useful and reliable results, none
is free from limitations.
Electrophoresis with demonstration of the pattern of isoenzymes zones by fluorescences is simple and rapid, and offers positive identification of the individual isoenzymes. However, this last advantage is somewhat offset changes in the mobility of BB-c.K. on storage or incubation of sera,
MB CREATINE KINASE DETECTION of myocardial damage, by increased activities in serum of intracellular enzymes such as creatine kinase (C.K.), aspartate transaminase (A.S.T.), and lactate dehydrogenase (L.D.), is one of the most widely used applications of diagnostic enzymology. The correlation between acute myocardial infarction and raised enzyme activity approaches 100% in correctly timed specimens for C.K. and A.S.T. However, all three enzymes are of wide distribution in the body so that, by themselves, the tests have little specificity for myocardial damage. The existence of certain enzymes in distinguishable isoenzymic forms with tissue-specific distributions can bring two advantages. The first is the ability to trace a raised enzyme activity in serum to its origin in a particular organ or tissue. The second is increased sensitivity-in that the distribution of isoenzymes in the serum may be abnormal in disease although the total activity of the enzyme is within normal limits. Both these advantages have already been exploited in cardiology with the isoenzymes of L.D., but the spotlight is now on the MB isoenzyme of C.K.’ Although cardiac muscle is not the exclusive source of this isoenzyme, measurement of MB-c.K. is the most sensitive enzymatic index of myocardial damage yet discovered. However, the successful diagnostic use of MB-c.K. depends on the availability of suitable analytical methods, which must be capable of detecting and measuring specifically the MB isoenzyme in the invariable presence of an excess of the MM isoenzyme and the possible presence of a substantial amount of the BB form. In general, isoenzyme analysis makes use of differences in catalytic properties—e.g., reactivity towards or affinity for substrate analogues or specific activators or inhibitors-or in physicochemical properties such as resistance to inactivation, net molecular charge, or antigenic specificity. Catalytic differences between C.K. isoenzymes are not great enough to form the basis of useful analytical methods, although differences in inhibition by excess substrate2 and, in particular, the selective effects of activators3 have attracted attention. Selective activation methods have not been uniformly 4. Hansard, Jan. 12, 1978, col. 807. 1. Roberts, R., Parker, L. W., Sobel, B. E. Lancet, 1977, ii, 319; and see correspondence, ibid. pp. 654, 759, 1127, 1283. 2. Witteveen, S. A. G. J., Sobel, B. E., DeLuca, M. Proc. Natn. Acad. Sci.
U.S.A. 1974, 71, 1384. 3. Rao, P. S., Lukes, J. J., Ayres, S. M., Mueller, H. Clin. Chem. 1975, 21, 1612.
so
that it
comes to
resemble the MB
isoenzyme.Several investigators believe that detection electrophoresis of serum allows a of positive diagnosis infarction, but with more sensitive procedures an MB zone may be detectable in non-infarctive conditions. Therefore, quantitation of the electrophoretic pattern is desirable, either by fluorimetric scanning or by measurement of the activity of eluted zones. Batchwise and stepwise ion-exchange chromatography’ are simple quantitative separation procedures. With these techniques, a possible source of error is failure to elute completely the excess of MM isoenzyme, part of which may be carried over into the MB isoenzyme fraction, so that the latter is overestimated. Immunochemical procedures which make use of inhibiting or precipitating antisera8-10 to the M subunit and which therefore cross-react with the hybrid MB isoenzyme but not with BB-c.K. can be combined with automated methods of determining C.K. activity. Radioimmunoassay (R.I.A.)l,l1,12 is potentially highly sensitive but takes longer. R.LA. will also measure antigenically intact but catalytically inactive isoenzymes, and therefore may increase the sensitivity of detection of tissue damage; however, preliminary results suggest that the time-courses of serum MB-c.K. changes after myocardial infarction are similar whether determined by R.I.A. or by catalytic-activity methods.u A major disadvantage of sole reliance on these immunochemical methods is that all B subunits are measured, whether present as MB or as BB isoenzymes. Although in many cases of suspected myocardial infarction B subunits are likely to be present only as components of MB-c.K. molecules, this may not invariably be so.There are several reports of BB-c.K . in serum, some in patients who have had cardiac operations. 13I Since all these analytical methods are expensive of time, skill, and reagents, what are the clinical indications for measurement? In most cases of uncomplicated myocardial infarction with characteristic electrocardioof
an
MB band after
Balkom, R. M. ibid. 1976, 22, 929. 5. Somer, H., Konttinen, A. Clinica chim. Acta. 1972, 40, 133. 6. Cho, H. W., Meltzer, H. Y., Joung, J. I., Goode, D. ibid. 1976, 73, 257. 7. Mercer, D. W. Clin. Chem. 1974, 20, 36. 8. Jockers-Wretou, E., Pfleiderer, G. Clinica chim. Acta. 1975, 58, 223. 9. Würzbarg, U., Hennrich, N., Lang, H., Prellwitz, W., Neumeier, D., Knedel, M. Klin. Wschr. 1976, 54, 357. 10. Gerhardt, W., Ljungdahl, L., Börjesson, J., Hofvendahl, S., Hedenäs, B. Clinica chim. Acta. 1977, 78, 43. 11. Roberts, R., Sobel, B. E., Parker, C. W. Science, 1976, 194, 855. 12. Willerson, J. T., Stone, M. J., Ting, R., Mukherjee, A., Gomez-Sanchez, C. E., Lewis, P., Hersch, L. B. Proc. natn. Acad. Sci., U.S. A. 1977, 74, 1711. 13. Vladutiu, A. O., Schachner, A., Schaeffer, P. A., Schimert, G., Lajos, T. Z., Lee, A. B., Siegel, J. H. Clinica chim. Acta. 1977, 75, 467. 4.
314
graphic changes and raised A.s.T. or C.K. in appropriate specimens of blood, isoenzyme analysis is not needed. The earlier or more conclusive evidence of myocardial infarction offered by MB-c.K. determinations may sometimes influence immediate or long-term treatment; but some methods are not sensitive enough to rule out infarction in the first few hours. The specificity of MB-c.K. for myocardial damage is most useful in the investigation of suspected myocardial infarction in patients in whom other enzymatic indices of myocardial damage are already compromised. This is the case, for example, in the postoperative period, when enzyme activities in serum are typically raised due to trauma and hypoxia of tissues, but when the risks of intra-operative or postoperative infarction are high, particularly in cardiac surgery. In such patients a more than transient rise in MB-c.K. in serum probably indicates myocardial infarction.14
A NEW ANTIMICROBIAL COMPOUND—Ro03-7008
MANIPULATION of molecular structure to yield an antimicrobial agent capable of incorporation into bacterial cells is a new approach to research into chemotherapeutic drugs. At the 10th International Congress of Chemotherapy, in Zurich, members of the scientific staff of Roche Products U.K. presented the first reports on the new antimicrobial agent ’Ro03-7008’ (S-alanyl-R-1aminoethyl-phosphonic acid), a peptide with potent inhibitory activity on cell-wall biosynthesis. Evaluation of the antibacterial properties of a wide range of dipeptides to pentapeptides, rationally designed as inhibitors of cell-wall biosynthesis, led to the selection of Ro03-7008 for clinical trials. The drug exploits active transport mechanisms in the cell membrane, being taken into the bacterium by specific permeases. Once it is inside the cell, hydrolysis takes place, yielding an alanine analogue capable of inhibiting two enzymes involved in the early stages of peptidoglycan synthesis. The bacterial cell wall is composed of glycan strands linked in layers by short peptide chains, which confer rigidity. The creation of these short peptide links is the first stage of cell-wall biosynthesis, and starts with the conversion of natural L-alanine to D-alanine, the linkage of two D-alanine molecules, and addition of further aminoacids.2 The alanine analogue derived from hydrolysis of Ro03-7008 is active at this early stage, inhibiting two enzymes, alanine racemase and alanyl:u.D.P.-muramyl ligase. It also acts as a false substrate, preventing further extension of the peptide chain; the result is disruption of the wall and cell lysis. The mode of action of cycloserine is similar; this drug is also a close alanine analogue, acting on the early stages of peptidoglycan. synthesis (in contrast to most other cell-wall antibiotics which interfere with later stages of bacterial cell-wall formation). Ro03-7008 has a moderately broad spectrum of activity, being preferentially active against gram-negatives, Dixon, S. H., Limbird, L. E., Roe, C. R., Wagner, G. S., Oldham, H. N., Sabiston, D. C. Circulation, 1973, 47, Suppl. 3, p. 137. 1. 10th International Congress of Chemotherapy, Zurich, 1977, abstracts 365,
14.
366 and 367. 2.
Garrod,
L.
P., Lamberts, H. P., O’Grady,
Edinburgh, 1973.
F. Antibiotic and
Chemotherapy.
but with bactericidal effects on certain gram-positive organisms. It has potent activity against E. coli, Kleb-
siella, Enterobacter, Providencia, Citrobacter, and
inhibitory at higher concentrations Haemophilus, Salmonella, Shigella, Staphylococcus, and Micrococcus species. The only common urinarytract pathogens not effectively inhibited are Proteus mirabilis and Pseudomonas ceruginosa. Proteus, because of its greater internal osmolarity, has a tendency to form protoplasts, in contrast to E. coli which undergoes rapid lysis. There is no cross-resistance pattern with other marketed antibiotics. Ro03-7008 is resistant to P4actaEnterococcus and is to
with other cell-wall antibiois similar to that of behaviour pharmacokinetic and with which this agent may penicillin cephalosporins, in useful combination. Resistance prove may emerge, gradually, since a slow, step-wise decrease in susceptibility has been observed in vitro. Antagonist-free medium is needed for sensitivity tests, because the presence of alanine and phosphopeptidases inhibits the activity of the drug. Similar problems have already been encountered in the sensitivity testing of organisms to sulphonamides and cycloserine; the need for special media in routine laboratories is a drawback. Rapid absorption follows intramuscular injection, and levels decline within a half-life of about one hour in man. The drug is excreted mainly by the kidney, but it is metabolised too; 20-30% of an intramuscular dose is excreted in urine as the original compound. The drug is well absorbed after oral administration and has been well tolerated in volunteers. It is not incorporated into mammalian protein and toxicity studies in rodents suggest a considerable margin of safety for man. Preliminary trials in patients with urinary-tract infection are in progress. mases, and shows synergy
tics ; the
THE DOCTOR’S ROLE IN RURAL HEALTH
THE troubles of rural communities, barely existing on subsistence economies, have been with us ever since man first settled to agriculture. The latest restatement of them is a document from the Royal Society, Technologies for Rural Health,! the report of a discussion meeting attended by doctors, biologists, economists, and other scientific experts. S. G. Browne begins by summarising the problems: the burgeoning towns, with their intolerable growth of slums, are taking up money, doctors, and curative services which are then denied to people in rural areas; food production lags behind the growth of population; bringing clean water to, and taking soiled water from, households would be the most important hygienic innovation conceivable; communicable disease should be controlled by immunisation, by chemoprophylaxis, or (in increasingly exceptional circumstances) by vector control. Much of this reads like a colonial medical report published in any year up to 1950, or a report to his rural district council by any medical officer of health in England before 1939. What then is new? Not the realisation that the world’s food supplies depend on rural health and that food production, though increasing, cannot keep pace with the 1.
Technologies for Rural Health: report of a Royal Society Discussion Meeting held on Dec. 9 and 10, 1976. The Royal Society (6 Carlton House Terrace, London SW1Y SAG): 1977. Pp. 187. £6·25 (U.K.), £6·45 overseas, incl. post and packing.
evident reasons for caution, until more facts have been gathered and assessed. Among the unanswered questions is why the reported total of children born alive with spina bifida in England and Wales has fallen from 1138 in 1970 to 678 in 19764-without routine screening. Particularly needed are better figures by which to compare the number of expected live spinabifida births with the number of normal fetuses lost either by accidental abortion after amniocentesis or by termination induced because of deceptive a-fetoprotein levels. But there
are
reliable.4 The distinct differences in net charge at a given pH result in good resolution of mixtures of C.K.
isoenzymes by zone electrophoresis or ion-exchange chromatography. Both these methods are widely used. C.K. isoenzymes, or more precisely their constituent M and B subunits, are antigenically distinct, and various immunochemical procedures have been applied to the study of C.K. isoenzymes in serum. Although all these methods are capable of giving useful and reliable results, none
is free from limitations.
Electrophoresis with demonstration of the pattern of isoenzymes zones by fluorescences is simple and rapid, and offers positive identification of the individual isoenzymes. However, this last advantage is somewhat offset changes in the mobility of BB-c.K. on storage or incubation of sera,
MB CREATINE KINASE DETECTION of myocardial damage, by increased activities in serum of intracellular enzymes such as creatine kinase (C.K.), aspartate transaminase (A.S.T.), and lactate dehydrogenase (L.D.), is one of the most widely used applications of diagnostic enzymology. The correlation between acute myocardial infarction and raised enzyme activity approaches 100% in correctly timed specimens for C.K. and A.S.T. However, all three enzymes are of wide distribution in the body so that, by themselves, the tests have little specificity for myocardial damage. The existence of certain enzymes in distinguishable isoenzymic forms with tissue-specific distributions can bring two advantages. The first is the ability to trace a raised enzyme activity in serum to its origin in a particular organ or tissue. The second is increased sensitivity-in that the distribution of isoenzymes in the serum may be abnormal in disease although the total activity of the enzyme is within normal limits. Both these advantages have already been exploited in cardiology with the isoenzymes of L.D., but the spotlight is now on the MB isoenzyme of C.K.’ Although cardiac muscle is not the exclusive source of this isoenzyme, measurement of MB-c.K. is the most sensitive enzymatic index of myocardial damage yet discovered. However, the successful diagnostic use of MB-c.K. depends on the availability of suitable analytical methods, which must be capable of detecting and measuring specifically the MB isoenzyme in the invariable presence of an excess of the MM isoenzyme and the possible presence of a substantial amount of the BB form. In general, isoenzyme analysis makes use of differences in catalytic properties—e.g., reactivity towards or affinity for substrate analogues or specific activators or inhibitors-or in physicochemical properties such as resistance to inactivation, net molecular charge, or antigenic specificity. Catalytic differences between C.K. isoenzymes are not great enough to form the basis of useful analytical methods, although differences in inhibition by excess substrate2 and, in particular, the selective effects of activators3 have attracted attention. Selective activation methods have not been uniformly 4. Hansard, Jan. 12, 1978, col. 807. 1. Roberts, R., Parker, L. W., Sobel, B. E. Lancet, 1977, ii, 319; and see correspondence, ibid. pp. 654, 759, 1127, 1283. 2. Witteveen, S. A. G. J., Sobel, B. E., DeLuca, M. Proc. Natn. Acad. Sci.
U.S.A. 1974, 71, 1384. 3. Rao, P. S., Lukes, J. J., Ayres, S. M., Mueller, H. Clin. Chem. 1975, 21, 1612.
so
that it
comes to
resemble the MB
isoenzyme.Several investigators believe that detection electrophoresis of serum allows a of positive diagnosis infarction, but with more sensitive procedures an MB zone may be detectable in non-infarctive conditions. Therefore, quantitation of the electrophoretic pattern is desirable, either by fluorimetric scanning or by measurement of the activity of eluted zones. Batchwise and stepwise ion-exchange chromatography’ are simple quantitative separation procedures. With these techniques, a possible source of error is failure to elute completely the excess of MM isoenzyme, part of which may be carried over into the MB isoenzyme fraction, so that the latter is overestimated. Immunochemical procedures which make use of inhibiting or precipitating antisera8-10 to the M subunit and which therefore cross-react with the hybrid MB isoenzyme but not with BB-c.K. can be combined with automated methods of determining C.K. activity. Radioimmunoassay (R.I.A.)l,l1,12 is potentially highly sensitive but takes longer. R.LA. will also measure antigenically intact but catalytically inactive isoenzymes, and therefore may increase the sensitivity of detection of tissue damage; however, preliminary results suggest that the time-courses of serum MB-c.K. changes after myocardial infarction are similar whether determined by R.I.A. or by catalytic-activity methods.u A major disadvantage of sole reliance on these immunochemical methods is that all B subunits are measured, whether present as MB or as BB isoenzymes. Although in many cases of suspected myocardial infarction B subunits are likely to be present only as components of MB-c.K. molecules, this may not invariably be so.There are several reports of BB-c.K . in serum, some in patients who have had cardiac operations. 13I Since all these analytical methods are expensive of time, skill, and reagents, what are the clinical indications for measurement? In most cases of uncomplicated myocardial infarction with characteristic electrocardioof
an
MB band after
Balkom, R. M. ibid. 1976, 22, 929. 5. Somer, H., Konttinen, A. Clinica chim. Acta. 1972, 40, 133. 6. Cho, H. W., Meltzer, H. Y., Joung, J. I., Goode, D. ibid. 1976, 73, 257. 7. Mercer, D. W. Clin. Chem. 1974, 20, 36. 8. Jockers-Wretou, E., Pfleiderer, G. Clinica chim. Acta. 1975, 58, 223. 9. Würzbarg, U., Hennrich, N., Lang, H., Prellwitz, W., Neumeier, D., Knedel, M. Klin. Wschr. 1976, 54, 357. 10. Gerhardt, W., Ljungdahl, L., Börjesson, J., Hofvendahl, S., Hedenäs, B. Clinica chim. Acta. 1977, 78, 43. 11. Roberts, R., Sobel, B. E., Parker, C. W. Science, 1976, 194, 855. 12. Willerson, J. T., Stone, M. J., Ting, R., Mukherjee, A., Gomez-Sanchez, C. E., Lewis, P., Hersch, L. B. Proc. natn. Acad. Sci., U.S. A. 1977, 74, 1711. 13. Vladutiu, A. O., Schachner, A., Schaeffer, P. A., Schimert, G., Lajos, T. Z., Lee, A. B., Siegel, J. H. Clinica chim. Acta. 1977, 75, 467. 4.
314
graphic changes and raised A.s.T. or C.K. in appropriate specimens of blood, isoenzyme analysis is not needed. The earlier or more conclusive evidence of myocardial infarction offered by MB-c.K. determinations may sometimes influence immediate or long-term treatment; but some methods are not sensitive enough to rule out infarction in the first few hours. The specificity of MB-c.K. for myocardial damage is most useful in the investigation of suspected myocardial infarction in patients in whom other enzymatic indices of myocardial damage are already compromised. This is the case, for example, in the postoperative period, when enzyme activities in serum are typically raised due to trauma and hypoxia of tissues, but when the risks of intra-operative or postoperative infarction are high, particularly in cardiac surgery. In such patients a more than transient rise in MB-c.K. in serum probably indicates myocardial infarction.14
A NEW ANTIMICROBIAL COMPOUND—Ro03-7008
MANIPULATION of molecular structure to yield an antimicrobial agent capable of incorporation into bacterial cells is a new approach to research into chemotherapeutic drugs. At the 10th International Congress of Chemotherapy, in Zurich, members of the scientific staff of Roche Products U.K. presented the first reports on the new antimicrobial agent ’Ro03-7008’ (S-alanyl-R-1aminoethyl-phosphonic acid), a peptide with potent inhibitory activity on cell-wall biosynthesis. Evaluation of the antibacterial properties of a wide range of dipeptides to pentapeptides, rationally designed as inhibitors of cell-wall biosynthesis, led to the selection of Ro03-7008 for clinical trials. The drug exploits active transport mechanisms in the cell membrane, being taken into the bacterium by specific permeases. Once it is inside the cell, hydrolysis takes place, yielding an alanine analogue capable of inhibiting two enzymes involved in the early stages of peptidoglycan synthesis. The bacterial cell wall is composed of glycan strands linked in layers by short peptide chains, which confer rigidity. The creation of these short peptide links is the first stage of cell-wall biosynthesis, and starts with the conversion of natural L-alanine to D-alanine, the linkage of two D-alanine molecules, and addition of further aminoacids.2 The alanine analogue derived from hydrolysis of Ro03-7008 is active at this early stage, inhibiting two enzymes, alanine racemase and alanyl:u.D.P.-muramyl ligase. It also acts as a false substrate, preventing further extension of the peptide chain; the result is disruption of the wall and cell lysis. The mode of action of cycloserine is similar; this drug is also a close alanine analogue, acting on the early stages of peptidoglycan. synthesis (in contrast to most other cell-wall antibiotics which interfere with later stages of bacterial cell-wall formation). Ro03-7008 has a moderately broad spectrum of activity, being preferentially active against gram-negatives, Dixon, S. H., Limbird, L. E., Roe, C. R., Wagner, G. S., Oldham, H. N., Sabiston, D. C. Circulation, 1973, 47, Suppl. 3, p. 137. 1. 10th International Congress of Chemotherapy, Zurich, 1977, abstracts 365,
14.
366 and 367. 2.
Garrod,
L.
P., Lamberts, H. P., O’Grady,
Edinburgh, 1973.
F. Antibiotic and
Chemotherapy.
but with bactericidal effects on certain gram-positive organisms. It has potent activity against E. coli, Kleb-
siella, Enterobacter, Providencia, Citrobacter, and
inhibitory at higher concentrations Haemophilus, Salmonella, Shigella, Staphylococcus, and Micrococcus species. The only common urinarytract pathogens not effectively inhibited are Proteus mirabilis and Pseudomonas ceruginosa. Proteus, because of its greater internal osmolarity, has a tendency to form protoplasts, in contrast to E. coli which undergoes rapid lysis. There is no cross-resistance pattern with other marketed antibiotics. Ro03-7008 is resistant to P4actaEnterococcus and is to
with other cell-wall antibiois similar to that of behaviour pharmacokinetic and with which this agent may penicillin cephalosporins, in useful combination. Resistance prove may emerge, gradually, since a slow, step-wise decrease in susceptibility has been observed in vitro. Antagonist-free medium is needed for sensitivity tests, because the presence of alanine and phosphopeptidases inhibits the activity of the drug. Similar problems have already been encountered in the sensitivity testing of organisms to sulphonamides and cycloserine; the need for special media in routine laboratories is a drawback. Rapid absorption follows intramuscular injection, and levels decline within a half-life of about one hour in man. The drug is excreted mainly by the kidney, but it is metabolised too; 20-30% of an intramuscular dose is excreted in urine as the original compound. The drug is well absorbed after oral administration and has been well tolerated in volunteers. It is not incorporated into mammalian protein and toxicity studies in rodents suggest a considerable margin of safety for man. Preliminary trials in patients with urinary-tract infection are in progress. mases, and shows synergy
tics ; the
THE DOCTOR’S ROLE IN RURAL HEALTH
THE troubles of rural communities, barely existing on subsistence economies, have been with us ever since man first settled to agriculture. The latest restatement of them is a document from the Royal Society, Technologies for Rural Health,! the report of a discussion meeting attended by doctors, biologists, economists, and other scientific experts. S. G. Browne begins by summarising the problems: the burgeoning towns, with their intolerable growth of slums, are taking up money, doctors, and curative services which are then denied to people in rural areas; food production lags behind the growth of population; bringing clean water to, and taking soiled water from, households would be the most important hygienic innovation conceivable; communicable disease should be controlled by immunisation, by chemoprophylaxis, or (in increasingly exceptional circumstances) by vector control. Much of this reads like a colonial medical report published in any year up to 1950, or a report to his rural district council by any medical officer of health in England before 1939. What then is new? Not the realisation that the world’s food supplies depend on rural health and that food production, though increasing, cannot keep pace with the 1.
Technologies for Rural Health: report of a Royal Society Discussion Meeting held on Dec. 9 and 10, 1976. The Royal Society (6 Carlton House Terrace, London SW1Y SAG): 1977. Pp. 187. £6·25 (U.K.), £6·45 overseas, incl. post and packing.
