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PLASMA-INSULIN IN MILD JUVENILE DIABETES
1257
lasted for six to eight hours) there was a gradual fall in the quantity of hepatic bile produced and in the insulin concentration of this bile. Since bile-salts in the bowel are an important stimulus to bile production by the liver, the reduction in flow was probably due to the continuous loss of bile-salts from our catheter. A sample of human gallbladder bile, taken eight hours post mortem from a woman with no gallbladder disease, showed an insulin concentration of 1300 U per ml. The tables show that there is virtually no insulin in c.s.F. and very little in aqueous humour from the anterior chamber of the eye.
The insulin levels in
lymph possibly
blood and thoracic duct in the rabbit than in the monkey, slightly higher because the monkeys were fasted and the rabbits venous
are
were not. DISCUSSION
PLASMA-INSULIN IN MILD JUVENILE DIABETES
fasting plasma-insulin levels and the response to various stimuli in three patients with mild juvenile diabetes suggested an abnormal regulation of insulin secretion as well as peripheral insulin resistance resembling that found in older patients. Summary
The
INTRODUCTION
SPONTANEOUS diabetes in man appears as two different clinical types, perhaps two different diseases: juvenile diabetes and late-onset diabetes. The clinical symptoms of juvenile diabetes set in acutely-usually the patient is able to give the week, or at least the month, in which the disease started. Juvenile diabetes is characterised by severe carbohydrate intolerance and a pronounced tendency to ketosis, and insulin treatment is obligatory. Late-onset diabetes develops gradually. There is a moderate but unquestionable rise of the blood-sugar level throughout the day, but symptoms are few or go unrecognised ; there is no ketosis; and there is no need for insulin treatment. Late-onset diabetes occurs commonly in older people, but in some old people diabetes may be severe, resembling juvenile diabetes in all respects. Children and young people have juvenile diabetes, as the term implies. Mildi.e., late-onset-diabetes is generally regarded as very uncommon in children and young persons, although a few cases have been described.1 2 Minor abnormalities of the glucose-tolerance curve do, of course, occur also in young
The extraordinarily high level of insulin in gallbladder bile prompts two questions: (1) is the substance present really insulin? and, if so (2) has it any physiological function ? We have begun to answer the first question by dilution studies: different dilutions of gallbladder bile have been assayed for their insulin concentration. In all the gallbladder samples the radioactivity of the boundinsulin complex corresponded well with the dilution level of gallbladder bile. The dilution-curves derived from two such series are shown in the accompanying figure. Satisfactory dilution-curves were also obtained with specimens of human and ox (slaughterhouse) gallbladder bile. These results strongly suggest that the substance in the gallbladder is insulin. Further work is in progress to isolate the insulin. people.34 Most constituents of hepatic bile are concentrated The following is a preliminary report on the plasma3-4 times in the gallbladder 6 so that the level of insulin insulin pattern, fasting and after various stimuli, in mild found in hepatic bile in these experiments, 30-35 U per juvenile diabetes mellitus. Plasma-insulin was determl., would be expected to produce a concentration of mined according to Hales and Randle 5 with addition of about 100 tU per ml. in gallbladder bile. Instead, the edetic acid (E.D.T.A.). The glucose-oxidase method was mean level we have found has been nearly 20 times this used for blood-sugar. predicted value, suggesting either that the gallbladder is CASE-REPORTS secreting insulin, or that some differential process of Case 1 reabsorption is occurring. This biliary insulin might A 19-year-old girl had no family history of diabetes. At the conceivably be of some value in absorption from the age of 15, glucosuria was discovered on routine examination intestine; on the other hand, if this insulin is merely a by the school doctor. She had never had any symptoms of waste
product-amounting
to
many milliunits
a
day-it
reckless extravagance of the body, in contrast to the parsimony with which other striking constituents of the bile are handled. It is tempting to relate the low insulin levels found in the aqueous humour of the anterior chamber of the eye with the high incidence of cataracts in human and experimental diabetes.’7 Cataracts are seen early in the overt diseases, possibly only a small reduction in circulating insulin would be necessary to deprive the anterior chamber of the insulin needed for the metabolism of the
would
seem
a
lens. We are grateful to the Nuffield Foundation and the Research Fund of the Bethlem Royal and Maudsley Hospitals for generous grants which assisted this work. J. R. H. is Lawrence research fellow of the British Diabetic Association. Request for reprints should be addressed to him.
Department
of Neuropathology, Institute of Psychiatry,
Maudsley Hospital, Denmark Hill, London S.E.5 6. Documenta
D.M.
P. M. DANIEL Oxon., D.SC. Lond., F.R.C.P. J. R. HENDERSON M.A., B.M.,
B.SC.
Oxon.
Geigy Scientific Tables; p. 525. Geigy Pharmaceuticals Ltd., Manchester, 1962. 7. Warren, S., LeCompte, P. M., Legg, M. A. The Pathology of Diabetes Mellitus; p. 247. London, 1966.
diabetes. The glucose-tolerance curve, however, was distinctly diabetic. Her blood-sugar, measured three times a day on several admissions, has been between 150 and 200 mg. per 100 ml.; but during her last admission the level was between 200 and 300 mg. per 100 ml. She was then put on a diet plus tolbutamide, 0-5 g. daily, which resulted in a normal level of the blood-sugar. She has never had ketosis. Her response pattern, determined several times, is shown in the accompanying figure. The glucose-tolerance tests show a plasma-insulin pattern similar to that of late-onset diabetes, and she responds to tolbutamide.
Case 2 A 20-year-old girl, whose parents and two other members of the family have diabetes, was found to have glucosuria incidentally by the school doctor when she was aged 15 years. Because she was thought to have mild symptoms of diabetes, she was put on a diet plus chlorpropamide. She stopped taking chlorpropamide after a year, but continued to avoid excess sugar. She felt perfectly well, but a year ago mild diabetes was diagnosed shortly before delivery of her first 1. Fajans, S. S., Conn, J. W. Diabetes, 1960, 9, 83. 2. Dolger, H. Proc. Congr. int. Diabetes, Fed. 1961; p. 356. 3. Br. med. J. 1963, ii, 655. 4. Sharp, C. L., Butterfield, W. J. H., Keen, H. Proc. R. Soc. Med. 57, 193. 5. Hales, C. N., Randle, P. J. Biochem. J. 1963, 88, 137.
1964,
1258 child. Her diurnal blood-sugar averaged about 200 mg. per 100 ml. After delivery she was put on a diet plus tolbutamide, 0-5 g. twice daily, and the 24-hour blood-sugar became normal. She has never had ketosis. Her response pattern (see figure) is similar to that seen in case 1. Case 3 A 15-year-old boy’s mother has diabetes. The school
doctor found glucosuria when the boy was aged 14. The boy had no diabetic symptoms, but his glucose-tolerance test was distinctly diabetic. On a normal diet, his blood-sugar was 100-200 mg. per 100 ml. throughout the day. Blood-sugar increased after the glucose-tolerance tests, but returned to a lower level after 1 week of moderate carbohydrate restriction. In January, 1967, his blood-sugar was found to be between
Plasma-insulin pattern in three different glucose-tolerance tests and tolbutamide test. Each point represents mean value ± standard error of mean from several tests.
1259 200 and 300 mg. per 100 ml. There were no signs of ketosis and no subjective symptoms. The boy was put on diet plus tolbutamide, but without effect. Blood-sugar was, however, restored to normal levels on phenformin, 50 mg. thrice daily. His response pattern was not quite the same as that of the other two patients, the plasma-insulin rising and remaining raised or decreasing during the course of the experiment (see figure). His response pattern, however, is quite different from that of the common variety of juvenile diabetes.
None of the
patients
was
obese.
DISCUSSION
The frequency and the natural history of mild juvenile diabetes are unknown. The disorder may be commoner than we think; it may represent an early phase of classical juvenile diabetes, or it could perhaps develop many years later into the common type of maturity-onset diabetes. The results presented here suggest an abnormal regulation of insulin secretion as well as peripheral insulin resistance, akin to the mild diabetes commonly found in old patients. Further studies are in progress to elucidate the frequency as well as the course of mild juvenile diabetes. KLAUS JOHANSEN Second Clinic of Internal Medicine,
Kommunehospitalet, Aarhus University School of Medicine, Aarhus, Denmark.
CAND. MED.
KNUD LUNDBÆK M.D.
Copenhagen
Hypothesis
gastric mucosa also appeared unable to attack these compounds. The decomposition of chloramphenicol had been earlier investigated by Smith and Worrell 6 7; chemical analysis enabled them to demonstrate breakage of the amide link by what they termed a chloramphenicolase produced by enterobacteria and Bacillus spp. They reported that some strains of the Bacillus and Proteus genera produced this enzyme in considerable quantities. Solid-state infra-red spectrography had proved valuable during investigations into the degradation of penicillin derivatives by bacterial enzymes 3 8; this method was therefore used in the investigation of chloramphenicol degradation. "
"
METHOD
Chloramphenicol at concentrations of 100 f1.g. per ml. and 500 f1.g. per ml. was incubated for 6 hours at 37°C with heavy suspensions of E. coli in 0-OlAf phosphate buffer at pH 6-0. The bacteria were then removed by high-speed centrifuge, and the supernatant was processed for infra-red studies by the potassium-bromide-micropellet technique described by Holt and Stewart.8 A small proportion of the supernatant was also assayed microbiologically to establish the degree of inactivation of antibacterial activity. In addition, liquid ultraviolet spectrography was used for evidence of possible structural changes in the chloramphenicol molecule. This series of tests was carried out by Miss E. M. Tanner,’who also repeated the infra-red spectrography on an instrument of greater sensitivity, and whose advice on the interpretation of the spectra was invaluable. RESULTS
THE BACTERIAL DEGRADATION OF CHLORAMPHENICOL THE degradation of certain antibacterial compounds with total loss of antibacterial activity by extracellular bacterial enzymes can be readily demonstrated by the cellulose-acetate-membrane method.1 This method was used originally to investigate this kind of activity towards derivatives of 6-aminopenicillanic acid and 7-aminocephalosporanic acid; later, and with modifications,2 a variety of organisms of different genera were tested against all the antibiotics in common use. The concentrations of the antibiotics in the base medium were in each case 1, 10, and 100 ug. per ml. Chloramphenicol and the polypeptide antibiotics polymyxin B and colistin appeared to be the only compounds attacked; all three were totally inactivated at each concentration by a few strains of Escherichia coli, freshly isolated from fasces. These were strains of the kind reported by us to produce large amounts of penicillinamidase active against the peptide linkage of several 6-aminopenicillanic acid derivatives. Hamilton-Miller 4 has given good reasons for calling this enzyme penicillin acylase. Cole and Sutherlandwere unable to confirm our views on the activity of these organisms against penicillin derivatives; working with different methods on some of our stored cultures, they concluded that this degradation was due to P-lactamase activity. However, many other strains of enterobacteria, found by us to destroy many 6-aminopenicillanic derivatives completely and rapidly, and therefore regarded as being prolific in P-lactamase production, showed little or no degradative action against chloramphenicol or the polypeptide antibiotics. A crude aminopeptidase preparation from pig 1. 2.
Holt, R. J., Stewart, G. T. J. clin. Path. 1963, 16, 263. Holt, R. J. 1967. Unpublished. 3. Holt, R. J., Stewart, G. T. J. gen. Microbiol. 1964, 36, 203. 4. Hamilton-Miller, J. M. T. Bact. Rev. 1966, 30, 761. 5. Cole, M., Sutherland, R. J. gen. Microbiol. 1966, 42, 345.
These studies revealed fractional changes in the 6-03 u band caused by the amide link in chloramphenicol, indicating the probability that chloramphenicol was under attack at this point. Alteration of the carbonyl band (5’95[jt.) of chloramphenicol could not be detected, since there was no separation of this band from that due to peptides in the bacterial enzyme also present. DISCUSSION
The risk of aplastic ansemia is now acknowledged to be inseparable from the administration of chloramphenicol, although this dyscrasia affects only a very small proportion of the patients treated.9 These people conceivably have in their gut a large population of enterobacteria capable of degrading chloramphenicol; the absorption of the byproducts of this action might thus lead to toxic depression of their marrow. Investigations in this hospital have been conducted almost entirely on children and, although exact figures are not available, apparently only a low proportion (probably under 2%) of these children have predominating gut flora of enterobacteria producing this enzyme prolifically. Significantly perhaps, there seems to be no recorded case in which marrow aplasia has followed administration of chloramphenicol by parenteral routes alone. This hypothesis could be tested by separating the breakdown products resulting from this enzymatic destruction of chloramphenicol and adding them individually and in combination to tissue-cultures of human bonemarrow ; the hope is that suitable tissue cultures will become available in this hospital. Chemical identification of the degradation products should be practicable-some were named by Smith and Worrell ’-and so would the synthesis in pure form of some of these compounds.10 6. Smith, G. N., Worrell, C. S. Archs Biochem. 1949, 24, 216. 7. Smith, G. N., Worrell, C. S. ibid. 1950, 28, 232. 8. Holt, R. J., Stewart, G. T. Biochem. biophys. Acta 1965, 100, 235. 9. Committee on Safety of Drugs. Chloramphenicol: Adverse Reactions, series no. 4. 1967. 10. Gottlieb, D. Personal communication. 1966.
lasted for six to eight hours) there was a gradual fall in the quantity of hepatic bile produced and in the insulin concentration of this bile. Since bile-salts in the bowel are an important stimulus to bile production by the liver, the reduction in flow was probably due to the continuous loss of bile-salts from our catheter. A sample of human gallbladder bile, taken eight hours post mortem from a woman with no gallbladder disease, showed an insulin concentration of 1300 U per ml. The tables show that there is virtually no insulin in c.s.F. and very little in aqueous humour from the anterior chamber of the eye.
The insulin levels in
lymph possibly
blood and thoracic duct in the rabbit than in the monkey, slightly higher because the monkeys were fasted and the rabbits venous
are
were not. DISCUSSION
PLASMA-INSULIN IN MILD JUVENILE DIABETES
fasting plasma-insulin levels and the response to various stimuli in three patients with mild juvenile diabetes suggested an abnormal regulation of insulin secretion as well as peripheral insulin resistance resembling that found in older patients. Summary
The
INTRODUCTION
SPONTANEOUS diabetes in man appears as two different clinical types, perhaps two different diseases: juvenile diabetes and late-onset diabetes. The clinical symptoms of juvenile diabetes set in acutely-usually the patient is able to give the week, or at least the month, in which the disease started. Juvenile diabetes is characterised by severe carbohydrate intolerance and a pronounced tendency to ketosis, and insulin treatment is obligatory. Late-onset diabetes develops gradually. There is a moderate but unquestionable rise of the blood-sugar level throughout the day, but symptoms are few or go unrecognised ; there is no ketosis; and there is no need for insulin treatment. Late-onset diabetes occurs commonly in older people, but in some old people diabetes may be severe, resembling juvenile diabetes in all respects. Children and young people have juvenile diabetes, as the term implies. Mildi.e., late-onset-diabetes is generally regarded as very uncommon in children and young persons, although a few cases have been described.1 2 Minor abnormalities of the glucose-tolerance curve do, of course, occur also in young
The extraordinarily high level of insulin in gallbladder bile prompts two questions: (1) is the substance present really insulin? and, if so (2) has it any physiological function ? We have begun to answer the first question by dilution studies: different dilutions of gallbladder bile have been assayed for their insulin concentration. In all the gallbladder samples the radioactivity of the boundinsulin complex corresponded well with the dilution level of gallbladder bile. The dilution-curves derived from two such series are shown in the accompanying figure. Satisfactory dilution-curves were also obtained with specimens of human and ox (slaughterhouse) gallbladder bile. These results strongly suggest that the substance in the gallbladder is insulin. Further work is in progress to isolate the insulin. people.34 Most constituents of hepatic bile are concentrated The following is a preliminary report on the plasma3-4 times in the gallbladder 6 so that the level of insulin insulin pattern, fasting and after various stimuli, in mild found in hepatic bile in these experiments, 30-35 U per juvenile diabetes mellitus. Plasma-insulin was determl., would be expected to produce a concentration of mined according to Hales and Randle 5 with addition of about 100 tU per ml. in gallbladder bile. Instead, the edetic acid (E.D.T.A.). The glucose-oxidase method was mean level we have found has been nearly 20 times this used for blood-sugar. predicted value, suggesting either that the gallbladder is CASE-REPORTS secreting insulin, or that some differential process of Case 1 reabsorption is occurring. This biliary insulin might A 19-year-old girl had no family history of diabetes. At the conceivably be of some value in absorption from the age of 15, glucosuria was discovered on routine examination intestine; on the other hand, if this insulin is merely a by the school doctor. She had never had any symptoms of waste
product-amounting
to
many milliunits
a
day-it
reckless extravagance of the body, in contrast to the parsimony with which other striking constituents of the bile are handled. It is tempting to relate the low insulin levels found in the aqueous humour of the anterior chamber of the eye with the high incidence of cataracts in human and experimental diabetes.’7 Cataracts are seen early in the overt diseases, possibly only a small reduction in circulating insulin would be necessary to deprive the anterior chamber of the insulin needed for the metabolism of the
would
seem
a
lens. We are grateful to the Nuffield Foundation and the Research Fund of the Bethlem Royal and Maudsley Hospitals for generous grants which assisted this work. J. R. H. is Lawrence research fellow of the British Diabetic Association. Request for reprints should be addressed to him.
Department
of Neuropathology, Institute of Psychiatry,
Maudsley Hospital, Denmark Hill, London S.E.5 6. Documenta
D.M.
P. M. DANIEL Oxon., D.SC. Lond., F.R.C.P. J. R. HENDERSON M.A., B.M.,
B.SC.
Oxon.
Geigy Scientific Tables; p. 525. Geigy Pharmaceuticals Ltd., Manchester, 1962. 7. Warren, S., LeCompte, P. M., Legg, M. A. The Pathology of Diabetes Mellitus; p. 247. London, 1966.
diabetes. The glucose-tolerance curve, however, was distinctly diabetic. Her blood-sugar, measured three times a day on several admissions, has been between 150 and 200 mg. per 100 ml.; but during her last admission the level was between 200 and 300 mg. per 100 ml. She was then put on a diet plus tolbutamide, 0-5 g. daily, which resulted in a normal level of the blood-sugar. She has never had ketosis. Her response pattern, determined several times, is shown in the accompanying figure. The glucose-tolerance tests show a plasma-insulin pattern similar to that of late-onset diabetes, and she responds to tolbutamide.
Case 2 A 20-year-old girl, whose parents and two other members of the family have diabetes, was found to have glucosuria incidentally by the school doctor when she was aged 15 years. Because she was thought to have mild symptoms of diabetes, she was put on a diet plus chlorpropamide. She stopped taking chlorpropamide after a year, but continued to avoid excess sugar. She felt perfectly well, but a year ago mild diabetes was diagnosed shortly before delivery of her first 1. Fajans, S. S., Conn, J. W. Diabetes, 1960, 9, 83. 2. Dolger, H. Proc. Congr. int. Diabetes, Fed. 1961; p. 356. 3. Br. med. J. 1963, ii, 655. 4. Sharp, C. L., Butterfield, W. J. H., Keen, H. Proc. R. Soc. Med. 57, 193. 5. Hales, C. N., Randle, P. J. Biochem. J. 1963, 88, 137.
1964,
1258 child. Her diurnal blood-sugar averaged about 200 mg. per 100 ml. After delivery she was put on a diet plus tolbutamide, 0-5 g. twice daily, and the 24-hour blood-sugar became normal. She has never had ketosis. Her response pattern (see figure) is similar to that seen in case 1. Case 3 A 15-year-old boy’s mother has diabetes. The school
doctor found glucosuria when the boy was aged 14. The boy had no diabetic symptoms, but his glucose-tolerance test was distinctly diabetic. On a normal diet, his blood-sugar was 100-200 mg. per 100 ml. throughout the day. Blood-sugar increased after the glucose-tolerance tests, but returned to a lower level after 1 week of moderate carbohydrate restriction. In January, 1967, his blood-sugar was found to be between
Plasma-insulin pattern in three different glucose-tolerance tests and tolbutamide test. Each point represents mean value ± standard error of mean from several tests.
1259 200 and 300 mg. per 100 ml. There were no signs of ketosis and no subjective symptoms. The boy was put on diet plus tolbutamide, but without effect. Blood-sugar was, however, restored to normal levels on phenformin, 50 mg. thrice daily. His response pattern was not quite the same as that of the other two patients, the plasma-insulin rising and remaining raised or decreasing during the course of the experiment (see figure). His response pattern, however, is quite different from that of the common variety of juvenile diabetes.
None of the
patients
was
obese.
DISCUSSION
The frequency and the natural history of mild juvenile diabetes are unknown. The disorder may be commoner than we think; it may represent an early phase of classical juvenile diabetes, or it could perhaps develop many years later into the common type of maturity-onset diabetes. The results presented here suggest an abnormal regulation of insulin secretion as well as peripheral insulin resistance, akin to the mild diabetes commonly found in old patients. Further studies are in progress to elucidate the frequency as well as the course of mild juvenile diabetes. KLAUS JOHANSEN Second Clinic of Internal Medicine,
Kommunehospitalet, Aarhus University School of Medicine, Aarhus, Denmark.
CAND. MED.
KNUD LUNDBÆK M.D.
Copenhagen
Hypothesis
gastric mucosa also appeared unable to attack these compounds. The decomposition of chloramphenicol had been earlier investigated by Smith and Worrell 6 7; chemical analysis enabled them to demonstrate breakage of the amide link by what they termed a chloramphenicolase produced by enterobacteria and Bacillus spp. They reported that some strains of the Bacillus and Proteus genera produced this enzyme in considerable quantities. Solid-state infra-red spectrography had proved valuable during investigations into the degradation of penicillin derivatives by bacterial enzymes 3 8; this method was therefore used in the investigation of chloramphenicol degradation. "
"
METHOD
Chloramphenicol at concentrations of 100 f1.g. per ml. and 500 f1.g. per ml. was incubated for 6 hours at 37°C with heavy suspensions of E. coli in 0-OlAf phosphate buffer at pH 6-0. The bacteria were then removed by high-speed centrifuge, and the supernatant was processed for infra-red studies by the potassium-bromide-micropellet technique described by Holt and Stewart.8 A small proportion of the supernatant was also assayed microbiologically to establish the degree of inactivation of antibacterial activity. In addition, liquid ultraviolet spectrography was used for evidence of possible structural changes in the chloramphenicol molecule. This series of tests was carried out by Miss E. M. Tanner,’who also repeated the infra-red spectrography on an instrument of greater sensitivity, and whose advice on the interpretation of the spectra was invaluable. RESULTS
THE BACTERIAL DEGRADATION OF CHLORAMPHENICOL THE degradation of certain antibacterial compounds with total loss of antibacterial activity by extracellular bacterial enzymes can be readily demonstrated by the cellulose-acetate-membrane method.1 This method was used originally to investigate this kind of activity towards derivatives of 6-aminopenicillanic acid and 7-aminocephalosporanic acid; later, and with modifications,2 a variety of organisms of different genera were tested against all the antibiotics in common use. The concentrations of the antibiotics in the base medium were in each case 1, 10, and 100 ug. per ml. Chloramphenicol and the polypeptide antibiotics polymyxin B and colistin appeared to be the only compounds attacked; all three were totally inactivated at each concentration by a few strains of Escherichia coli, freshly isolated from fasces. These were strains of the kind reported by us to produce large amounts of penicillinamidase active against the peptide linkage of several 6-aminopenicillanic acid derivatives. Hamilton-Miller 4 has given good reasons for calling this enzyme penicillin acylase. Cole and Sutherlandwere unable to confirm our views on the activity of these organisms against penicillin derivatives; working with different methods on some of our stored cultures, they concluded that this degradation was due to P-lactamase activity. However, many other strains of enterobacteria, found by us to destroy many 6-aminopenicillanic derivatives completely and rapidly, and therefore regarded as being prolific in P-lactamase production, showed little or no degradative action against chloramphenicol or the polypeptide antibiotics. A crude aminopeptidase preparation from pig 1. 2.
Holt, R. J., Stewart, G. T. J. clin. Path. 1963, 16, 263. Holt, R. J. 1967. Unpublished. 3. Holt, R. J., Stewart, G. T. J. gen. Microbiol. 1964, 36, 203. 4. Hamilton-Miller, J. M. T. Bact. Rev. 1966, 30, 761. 5. Cole, M., Sutherland, R. J. gen. Microbiol. 1966, 42, 345.
These studies revealed fractional changes in the 6-03 u band caused by the amide link in chloramphenicol, indicating the probability that chloramphenicol was under attack at this point. Alteration of the carbonyl band (5’95[jt.) of chloramphenicol could not be detected, since there was no separation of this band from that due to peptides in the bacterial enzyme also present. DISCUSSION
The risk of aplastic ansemia is now acknowledged to be inseparable from the administration of chloramphenicol, although this dyscrasia affects only a very small proportion of the patients treated.9 These people conceivably have in their gut a large population of enterobacteria capable of degrading chloramphenicol; the absorption of the byproducts of this action might thus lead to toxic depression of their marrow. Investigations in this hospital have been conducted almost entirely on children and, although exact figures are not available, apparently only a low proportion (probably under 2%) of these children have predominating gut flora of enterobacteria producing this enzyme prolifically. Significantly perhaps, there seems to be no recorded case in which marrow aplasia has followed administration of chloramphenicol by parenteral routes alone. This hypothesis could be tested by separating the breakdown products resulting from this enzymatic destruction of chloramphenicol and adding them individually and in combination to tissue-cultures of human bonemarrow ; the hope is that suitable tissue cultures will become available in this hospital. Chemical identification of the degradation products should be practicable-some were named by Smith and Worrell ’-and so would the synthesis in pure form of some of these compounds.10 6. Smith, G. N., Worrell, C. S. Archs Biochem. 1949, 24, 216. 7. Smith, G. N., Worrell, C. S. ibid. 1950, 28, 232. 8. Holt, R. J., Stewart, G. T. Biochem. biophys. Acta 1965, 100, 235. 9. Committee on Safety of Drugs. Chloramphenicol: Adverse Reactions, series no. 4. 1967. 10. Gottlieb, D. Personal communication. 1966.