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BARBITURATES GIVING ATYPICAL RESULTS ON ULTRAVIOLET SPECTROPHOTOMETRIC ANALYSIS
1266
patient is discharged prematurely in opposition to Cranbrook recommendations. There seems to be little logic in this. If a private patient, with a well-appointed home including all amenities and capital resources to buy services, requires a hospital lying-in period of 10 days, how much more so does the non-private patient who may well have come from an inadequate home with various sanitary defects and limited amenities ? ary
Public Health Office, Blackburn.
J. ARDLEY.
MERCURY AND THE KIDNEY
SIR,-We read your annotation (Feb. 2) and the discussion (Feb. 16) on mercury and the kidney with much interest. In the past three years we had 4 patients with proteinuria which we attributed to chronic mercury intoxication: in 1, occupational contact with mercury was the cause; 3 were patients with psoriasis which had been treated with mercurial ointments for long periods. 2 patients had the nephrotic syndrome, 2 had asymptomatic persistent proteinuria. Light microscopy in 3 of the patients showed only slight lesions of the glomeruli and some focal atrophy of renal tubuli; a renal biopsy-specimen in the 4th patient showed diffuse membranous changes. Electron microscopy of this biopsy specimen revealed that the thickening of the basement membrane was almost entirely caused by material which was laid down on the outside of the capillary wall, between the epithelial cells and the basement membrane proper. Electron microscopy was also done in 1 of the patients in whom light microscopy did not show definite abnormalities of the glomeruli. In this patient, there were a number of deposits of material between the epithelial cells and the basement membrane proper (fig. 1). Using the technique described Hardwick and Soothill, we found that the proteinuria was by " selective " in this patient and " non-selective " in the patient with diffuse membranous changes. 2 of the 4 patients received steroids; in both a rapid and heavy increase in protein excretion of short duration resulted (fig. 2). The patient with the diffuse membranous thickening still has persistent asymptomatic proteinuria; the other 3 recovered after contact with mercury ceased. The proteinuria cleared in 1 after three weeks; in the other 2 after five months and six months. Other symptoms of mercury poisoning were not observed, but we did not look for mercurialentis.
We think that when contact with mercury is stopped spontaneous recovery is the rule, with the possible exception of patients in whom diffuse severe membranous thickening has developed. Our patient with diffuse
Fig. 2-Effect of treatment with triamcinolone on proteinuria in a patient with psoriasis and chronic mercury intoxication (same patient as fig. 1).
membranous changes has been followed up for only six months after cessation of contact with mercury, so recovery is still possible. This work is supported by the Netherlands Organization for the Advance of Pure Research (Z.W.O.), FUNGO, group on proteins. E. MANDEMA A. ARENDS
J. Department of Medicine and Department of Pathology, University of Groningen, The Netherlands.
ZEIJST
G. VERMEER G. K. VAN DER HEM L. B. VAN DER SLIKKE.
BARBITURATES GIVING ATYPICAL RESULTS ON ULTRAVIOLET SPECTROPHOTOMETRIC ANALYSIS
SIR,-In their letter (April 20), Dr. George Matthews described some anomalous results when analysing barbiturates in the presence
and Miss obtained
of, other
drugs. In each instance the absorption spectra obtained were not characteristic of barbiturates,l but were sufficiently abnormal to indicate that some other compound was present, which might be masking the presence of barbiturate. In these circumstances, paper chromatographic methods 2-s for detecting barbiturates are not only invaluable but may also be used to identify any other drugs present. If the interfering material is known, relatively simple methods can often be devised for overcoming its effect on the spectrophotometric analysis of barbiturates. Interference by bemegride may be suspected when the sodium-hydroxide extract shows a disproportionately high extinction at wavelengths below 240 m!J.. Bemegride decomposes rapidly in strong alkali,l as is shown by a progressive decrease of extinction readings at 230 mpL, and may be completely destroyed by incubation at 38°C for 2 hours.Alkaline solutions of carbromal also absorb at wavelengths below 250 m, and decompose rapidly at pH 13. This interference may be found during gastric analysis in patients with ’Carbrital ’ (carbromal plus pentobarbitone) poisoning, but may be overcome by alkaline hydrolysis of the carbromal, which will not affect pentobarbitone. Other compounds which may interfere with 1. 2.
Fig. l-Electromicrograph of peripheral capillary loop of patient with chronic mercury intoxication. Note deposit of material between epithelial cell and basement membrane proper. ( 12,000.)
VAN
3. 4. 5. 6. 7.
Broughton, P. M. G. Biochem. J. 1956, 63, 207. Jackson, J. V., Moss, M. S. in Chromatographic Techniques (edited by Ivor Smith); vol. I. London, 1960. Street, H. V. Clin. chim. Acta, 1962, 7, 107. Street, H. V. Proc. Ass. clin. Biochem. 1962, 2, 44. Podmore, D. A. Clin. chim. Acta, 1962, 7, 176. Curry, A. S. in Toxicology: Mechanisms and Analytical Methods (edited by C. P. Stewart and A. Stolman); vol. II. New York, 1961. Curry, A. S. J. Pharm., Lond. 1957, 9, 102.
1267
ultraviolet spectrophotometric methods
are discussed by should be Their always suspected when presence Curry. obtained. are spectra atypical George and Matthews found that quinalbarbitone and amylobarbitone separately gave typical barbiturate spectra, but a combination of the two (’ Tuinal ’) gave curves with a " raised caustic component and an isosbestic point between 225 and 230 mp<. ". An isosbestic point in this range is one of the criteria used for the detection of barbiturates,l and in my experience the analysis of specimens from cases of tuinal poisoning has presented no unusual difficulties.
Department of Pathology, St. John’s Hospital,
P. M. G. BROUGHTON.
Chelmsford.
PHYSICALLY HANDICAPPED BABIES SIR,-Referring to the article of May 4 by Dr. White Franklin, this Association was a little disappointed that
he omitted the occupational therapist in his discussion of groups and teams. He seems to mention all other members of the team0—i.e., orthopædic surgeons, limb-
fitters, paediatricians, physiotherapists, educationists, aurists, and ophthalmologists, but there is no mention of
occupational therapists, and in our view they have quite a part to play in the treatment of these children. In several centres throughout the country, occupational therapists are already playing a large part in the treatment and
care
of these children.
B. B M. B. M. STOW
Chairman of Council, Association of Occupational Therapists.
London, S.W.3.
A TEST FOR BACTERIURIA
SIR,-Simmons and Williamsreported
simple, relatively rapid test for detecting significant bacteriuria, based on the ability of actively respiring bacteria to reduce at an alkaline pH the colourless soluble compound 2,3,5 triphenyl tetrazolium chloride (T.T.C.) to the red insoluble triphenyl formazan. According to these authors, a positive T.T.c. test (red precipitate) resulted when 0-5 ml. (150 -g.) of phosphate-buffered T.T.C. reagent was added to 2 ml. of urine containing 105 or more organisms per ml. and was incubated at 630C for on a
four hours. In view of the close correlation reported by the authors of the test, a study was made in this hospital comparing the T.T.C. test results and quantitative bacterial counting results obtained with 112 specimens of urine. The T.T.c. tests were performed according to Simmons and Williams, using three different lots of T.T.C. Two samples of each urine specimen were T.T.C. tested. One of each pair of samples was inoculated with 10cells per ml. of Escherichia coli as a control. All but 3 of the 112 control tests were T.T.c. positive. Simmons and Williams found a high degree of correlation (94%) between a positive T.T.c. test and a bacterial count of FAILURE OF T.T.C. TESTS TO CORRELATE CLOSELY WITH BACTERIAL COUNTS IN 112 SPECIMENS OF URINE
*
All 15 specimens had bacterial counts of > 10* per ml. t Four specimens had bacterial counts of > 10- per ml. 6 varied from >
10 to
>
10 per ml.
Remaining
106 or more organisms per ml. of urine specimen. Examination accompanying table, however, reveals a much lower percentage (60%) of true T.T.C. positives and therefore a higher percentage (40%) of false negative T.T.C. tests. Simmons and Williams reported 100% correlation of positive T.T.C. tests with the bacterial counts for urines infected of our data in the
8.
Simmons, N. A., Williams, J. G. Lancet, 1962, i,
1377.
with 105 or more organisms per ml. of a single species of gram-negative bacteria. 4 of their 7 false negatives contained mixed species and 3 were pure cultures of Streptococcusfacalis. 8 of our 10 false negatives listed in the table were pure cultures of gram-negative coliforms (6 Esch. coli, 1 Proteus vulgaris, and 1 Pseudomonas aeruginosa), and 2 were Str, fiecalis. All 15 true T.T.c. positives determined by us .contained more than 106 bacteria per ml. urine. Therefore, in order to determine the initial minimum inoculum of bacterial cells required per ml. of urine to yield a positive T.T.c. test within four hours incubation at 37°C, numerous T.T.C. tests were done using different urines (previously sterilised by filtration through UF sintered glass filters) which were inoculated with graded numbers of either Esch. coli, Aerobacter aerogenes, Staphylococcus aureus, or Str. fiecalis. Only those tests containing an initial inoculum of more than 106 cells per ml. were T.T.C. positive in four hours.
In view of our findings we question the value of the test, as described by Simmons and Williams, as screening test for detecting urinary-tract infections.
T.T.C. a
William Pepper Laboratory of Clinical Medicine, and Department of Medicine, School of Medicine, University of Pennsylvania,
Philadelphia.
EDWARD STEERS FRANK W. JACKSON,
II.
ANTIBIOTICS AND CONGENITAL MALFORMATIONS SIR,-Last year we reported the case of a baby with hand malformations born to a mother who had had tetracycline at a time in pregnancy exactly " corresponding "
with the defects observed.1 One of us (F. W.), looking through the medical records of pregnant women delivered in the previous year or two for other instances of mothers who had had tetracycline in early pregnancy, noticed that in 3 or 4 instances the administration to the mother of penicillin in early pregnancy had been followed by the birth of a malformed baby. We therefore analysed predetermined samples of recent deliveries in our respective general practices, and found among 387 pregnancies 13 where the mother had been prescribed an antibiotic in the first twelve weeks of pregnancy. Of these 13 mothers, 1 aborted, and no less than 6 had malformed babies; only 6 had normal babies. The incidence of malformations in mothers who did not have an antibiotic in the first twelve weeks was 3%, in contrast with the 46% malformations where the mother did have an antibiotic in early pregnancy. Since the " penicillin " cases originally noticed were included in the analysis the association reported above may be exaggerated, and it would be improper to apply formal tests of statistical significance to these figures. Nevertheless, we found them both striking and alarming. In those cases where the malformation was one which could be said to be induced at a definite period in pregnancy, the antibiotics appeared to have been given at the " right " time to induce the malformations observed. Filippi and Mela 2-4 produced malformations in the rat by administering antibiotics (tetracycline or a penicillinstreptomycin mixture) to pregnant rats, in doses calculated to be equivalent, weight for weight, to those used therapeutically in man. The simultaneous administration of vitamin-B complex to the rats prevented the malformations associated with tetracycline administration, but not those associated with the penicillin-streptomycin combination. Prof. A. Giroud5 has also obtained a malWilson, F. Brit. med. J. 1962, ii, 407. Filippi, B., Mela, V. Minerva Chir. 1957, 12, 1047. Filippi, B., Mela, V. ibid. p. 1106. Filippi, B., Mela, V. Arch. franc. Pédiat. 1958, 15, 565. Giroud, A. Personal communication, 1962.
1. Carter, M. P., 2. 3. 4. 5.
patient is discharged prematurely in opposition to Cranbrook recommendations. There seems to be little logic in this. If a private patient, with a well-appointed home including all amenities and capital resources to buy services, requires a hospital lying-in period of 10 days, how much more so does the non-private patient who may well have come from an inadequate home with various sanitary defects and limited amenities ? ary
Public Health Office, Blackburn.
J. ARDLEY.
MERCURY AND THE KIDNEY
SIR,-We read your annotation (Feb. 2) and the discussion (Feb. 16) on mercury and the kidney with much interest. In the past three years we had 4 patients with proteinuria which we attributed to chronic mercury intoxication: in 1, occupational contact with mercury was the cause; 3 were patients with psoriasis which had been treated with mercurial ointments for long periods. 2 patients had the nephrotic syndrome, 2 had asymptomatic persistent proteinuria. Light microscopy in 3 of the patients showed only slight lesions of the glomeruli and some focal atrophy of renal tubuli; a renal biopsy-specimen in the 4th patient showed diffuse membranous changes. Electron microscopy of this biopsy specimen revealed that the thickening of the basement membrane was almost entirely caused by material which was laid down on the outside of the capillary wall, between the epithelial cells and the basement membrane proper. Electron microscopy was also done in 1 of the patients in whom light microscopy did not show definite abnormalities of the glomeruli. In this patient, there were a number of deposits of material between the epithelial cells and the basement membrane proper (fig. 1). Using the technique described Hardwick and Soothill, we found that the proteinuria was by " selective " in this patient and " non-selective " in the patient with diffuse membranous changes. 2 of the 4 patients received steroids; in both a rapid and heavy increase in protein excretion of short duration resulted (fig. 2). The patient with the diffuse membranous thickening still has persistent asymptomatic proteinuria; the other 3 recovered after contact with mercury ceased. The proteinuria cleared in 1 after three weeks; in the other 2 after five months and six months. Other symptoms of mercury poisoning were not observed, but we did not look for mercurialentis.
We think that when contact with mercury is stopped spontaneous recovery is the rule, with the possible exception of patients in whom diffuse severe membranous thickening has developed. Our patient with diffuse
Fig. 2-Effect of treatment with triamcinolone on proteinuria in a patient with psoriasis and chronic mercury intoxication (same patient as fig. 1).
membranous changes has been followed up for only six months after cessation of contact with mercury, so recovery is still possible. This work is supported by the Netherlands Organization for the Advance of Pure Research (Z.W.O.), FUNGO, group on proteins. E. MANDEMA A. ARENDS
J. Department of Medicine and Department of Pathology, University of Groningen, The Netherlands.
ZEIJST
G. VERMEER G. K. VAN DER HEM L. B. VAN DER SLIKKE.
BARBITURATES GIVING ATYPICAL RESULTS ON ULTRAVIOLET SPECTROPHOTOMETRIC ANALYSIS
SIR,-In their letter (April 20), Dr. George Matthews described some anomalous results when analysing barbiturates in the presence
and Miss obtained
of, other
drugs. In each instance the absorption spectra obtained were not characteristic of barbiturates,l but were sufficiently abnormal to indicate that some other compound was present, which might be masking the presence of barbiturate. In these circumstances, paper chromatographic methods 2-s for detecting barbiturates are not only invaluable but may also be used to identify any other drugs present. If the interfering material is known, relatively simple methods can often be devised for overcoming its effect on the spectrophotometric analysis of barbiturates. Interference by bemegride may be suspected when the sodium-hydroxide extract shows a disproportionately high extinction at wavelengths below 240 m!J.. Bemegride decomposes rapidly in strong alkali,l as is shown by a progressive decrease of extinction readings at 230 mpL, and may be completely destroyed by incubation at 38°C for 2 hours.Alkaline solutions of carbromal also absorb at wavelengths below 250 m, and decompose rapidly at pH 13. This interference may be found during gastric analysis in patients with ’Carbrital ’ (carbromal plus pentobarbitone) poisoning, but may be overcome by alkaline hydrolysis of the carbromal, which will not affect pentobarbitone. Other compounds which may interfere with 1. 2.
Fig. l-Electromicrograph of peripheral capillary loop of patient with chronic mercury intoxication. Note deposit of material between epithelial cell and basement membrane proper. ( 12,000.)
VAN
3. 4. 5. 6. 7.
Broughton, P. M. G. Biochem. J. 1956, 63, 207. Jackson, J. V., Moss, M. S. in Chromatographic Techniques (edited by Ivor Smith); vol. I. London, 1960. Street, H. V. Clin. chim. Acta, 1962, 7, 107. Street, H. V. Proc. Ass. clin. Biochem. 1962, 2, 44. Podmore, D. A. Clin. chim. Acta, 1962, 7, 176. Curry, A. S. in Toxicology: Mechanisms and Analytical Methods (edited by C. P. Stewart and A. Stolman); vol. II. New York, 1961. Curry, A. S. J. Pharm., Lond. 1957, 9, 102.
1267
ultraviolet spectrophotometric methods
are discussed by should be Their always suspected when presence Curry. obtained. are spectra atypical George and Matthews found that quinalbarbitone and amylobarbitone separately gave typical barbiturate spectra, but a combination of the two (’ Tuinal ’) gave curves with a " raised caustic component and an isosbestic point between 225 and 230 mp<. ". An isosbestic point in this range is one of the criteria used for the detection of barbiturates,l and in my experience the analysis of specimens from cases of tuinal poisoning has presented no unusual difficulties.
Department of Pathology, St. John’s Hospital,
P. M. G. BROUGHTON.
Chelmsford.
PHYSICALLY HANDICAPPED BABIES SIR,-Referring to the article of May 4 by Dr. White Franklin, this Association was a little disappointed that
he omitted the occupational therapist in his discussion of groups and teams. He seems to mention all other members of the team0—i.e., orthopædic surgeons, limb-
fitters, paediatricians, physiotherapists, educationists, aurists, and ophthalmologists, but there is no mention of
occupational therapists, and in our view they have quite a part to play in the treatment of these children. In several centres throughout the country, occupational therapists are already playing a large part in the treatment and
care
of these children.
B. B M. B. M. STOW
Chairman of Council, Association of Occupational Therapists.
London, S.W.3.
A TEST FOR BACTERIURIA
SIR,-Simmons and Williamsreported
simple, relatively rapid test for detecting significant bacteriuria, based on the ability of actively respiring bacteria to reduce at an alkaline pH the colourless soluble compound 2,3,5 triphenyl tetrazolium chloride (T.T.C.) to the red insoluble triphenyl formazan. According to these authors, a positive T.T.c. test (red precipitate) resulted when 0-5 ml. (150 -g.) of phosphate-buffered T.T.C. reagent was added to 2 ml. of urine containing 105 or more organisms per ml. and was incubated at 630C for on a
four hours. In view of the close correlation reported by the authors of the test, a study was made in this hospital comparing the T.T.C. test results and quantitative bacterial counting results obtained with 112 specimens of urine. The T.T.c. tests were performed according to Simmons and Williams, using three different lots of T.T.C. Two samples of each urine specimen were T.T.C. tested. One of each pair of samples was inoculated with 10cells per ml. of Escherichia coli as a control. All but 3 of the 112 control tests were T.T.c. positive. Simmons and Williams found a high degree of correlation (94%) between a positive T.T.c. test and a bacterial count of FAILURE OF T.T.C. TESTS TO CORRELATE CLOSELY WITH BACTERIAL COUNTS IN 112 SPECIMENS OF URINE
*
All 15 specimens had bacterial counts of > 10* per ml. t Four specimens had bacterial counts of > 10- per ml. 6 varied from >
10 to
>
10 per ml.
Remaining
106 or more organisms per ml. of urine specimen. Examination accompanying table, however, reveals a much lower percentage (60%) of true T.T.C. positives and therefore a higher percentage (40%) of false negative T.T.C. tests. Simmons and Williams reported 100% correlation of positive T.T.C. tests with the bacterial counts for urines infected of our data in the
8.
Simmons, N. A., Williams, J. G. Lancet, 1962, i,
1377.
with 105 or more organisms per ml. of a single species of gram-negative bacteria. 4 of their 7 false negatives contained mixed species and 3 were pure cultures of Streptococcusfacalis. 8 of our 10 false negatives listed in the table were pure cultures of gram-negative coliforms (6 Esch. coli, 1 Proteus vulgaris, and 1 Pseudomonas aeruginosa), and 2 were Str, fiecalis. All 15 true T.T.c. positives determined by us .contained more than 106 bacteria per ml. urine. Therefore, in order to determine the initial minimum inoculum of bacterial cells required per ml. of urine to yield a positive T.T.c. test within four hours incubation at 37°C, numerous T.T.C. tests were done using different urines (previously sterilised by filtration through UF sintered glass filters) which were inoculated with graded numbers of either Esch. coli, Aerobacter aerogenes, Staphylococcus aureus, or Str. fiecalis. Only those tests containing an initial inoculum of more than 106 cells per ml. were T.T.C. positive in four hours.
In view of our findings we question the value of the test, as described by Simmons and Williams, as screening test for detecting urinary-tract infections.
T.T.C. a
William Pepper Laboratory of Clinical Medicine, and Department of Medicine, School of Medicine, University of Pennsylvania,
Philadelphia.
EDWARD STEERS FRANK W. JACKSON,
II.
ANTIBIOTICS AND CONGENITAL MALFORMATIONS SIR,-Last year we reported the case of a baby with hand malformations born to a mother who had had tetracycline at a time in pregnancy exactly " corresponding "
with the defects observed.1 One of us (F. W.), looking through the medical records of pregnant women delivered in the previous year or two for other instances of mothers who had had tetracycline in early pregnancy, noticed that in 3 or 4 instances the administration to the mother of penicillin in early pregnancy had been followed by the birth of a malformed baby. We therefore analysed predetermined samples of recent deliveries in our respective general practices, and found among 387 pregnancies 13 where the mother had been prescribed an antibiotic in the first twelve weeks of pregnancy. Of these 13 mothers, 1 aborted, and no less than 6 had malformed babies; only 6 had normal babies. The incidence of malformations in mothers who did not have an antibiotic in the first twelve weeks was 3%, in contrast with the 46% malformations where the mother did have an antibiotic in early pregnancy. Since the " penicillin " cases originally noticed were included in the analysis the association reported above may be exaggerated, and it would be improper to apply formal tests of statistical significance to these figures. Nevertheless, we found them both striking and alarming. In those cases where the malformation was one which could be said to be induced at a definite period in pregnancy, the antibiotics appeared to have been given at the " right " time to induce the malformations observed. Filippi and Mela 2-4 produced malformations in the rat by administering antibiotics (tetracycline or a penicillinstreptomycin mixture) to pregnant rats, in doses calculated to be equivalent, weight for weight, to those used therapeutically in man. The simultaneous administration of vitamin-B complex to the rats prevented the malformations associated with tetracycline administration, but not those associated with the penicillin-streptomycin combination. Prof. A. Giroud5 has also obtained a malWilson, F. Brit. med. J. 1962, ii, 407. Filippi, B., Mela, V. Minerva Chir. 1957, 12, 1047. Filippi, B., Mela, V. ibid. p. 1106. Filippi, B., Mela, V. Arch. franc. Pédiat. 1958, 15, 565. Giroud, A. Personal communication, 1962.
1. Carter, M. P., 2. 3. 4. 5.