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PYRUVATE KINASE IN HUMAN ERYTHROCYTES
529 TABLE
1.—PERCENTAGE
COMPOSITION
OF
PLATELET CHOLESTEROL, IN NORMAL SUBJECTS
TRIGLYCERIDES, AND TOTAL PHOSPHOLIPIDS AND MULTIPLE-SCLEROSIS PATIENTS
Each
figure represents
mean
of 5
experiments ::1=
s.E.
TABLE II.-PERCENTAGE COMPOSITION OF SINGLE PLATELET-PHOSPHOLIPID FRACTIONS OF NORMAL SUBJECTS AND MULTIPLE-SCLEROSIS PATIENTS
Numbers of
experiments
are
shown in
parentheses.
The results indicate that the differences in metabolic response and sensitivity to A.D.P. observed in platelets of M.S. patients are not due to changes of the major neutral lipids and phospholipids. Plasma alterations, as described by Dr. Bolton and his colleagues, may therefore be the important factor in platelet behaviour in patients with M.S., as indirectly confirmed by our results. However, the possibility that alterations in long-chain fatty-acid composition of phospholipids present in plateletmembranes may also play a relevant role should be considered. Investigations are being carried out in our laboratory along both these lines. V. M. ANDREOLI. Department of Pharmacology, Multiple Sclerosis Centre, University of Milan, Italy.
PYRUVATE KINASE IN HUMAN ERYTHROCYTES
SIR,-We report here a technique for the separation of pyruvate-kinase isozymes from human erythrocytes by electrophoresis on cellulose acetate in a gel form (’ Cellogel ’, Chemetron, Milan, Italy); cellogel has been used for investigating lactic dehydrogenase isozymes,1 glucose-6-phosphate dehydrogenase,2and glutathione-reductase isozymes.3 By high voltage electrophoresis at pH 62 two distinct isozymes can be observed, one band (PK I) moving towards the anode, the other (PK II) towards the cathode (see accompanying figure). In patients with increased enzyme activity 4 (A, 92 units per 1011 erythrocytes), which are associated with augmented adenosine-triphosphate-values in the erythrocytes, the PK I spot is larger than PK I of the usual enzyme (C, 40 units per 1011 erythrocytes). In patients with partial deficiency of pyruvate kinase (B, 16 units per 1011 erythrocytes), who clinically show a hamiolytic, non-spherocytic anxmia,5 the spot of PK I is smaller and moves more slowly from the origin. The mobility of the PK II isozymes seems to be similar in all three cases. In white blood-cells and platelets there is only one isozyme, which moves very slowly from the origin towards the cathode. For preparation of hoemolysates, the following mixture is used: erythrocytes (three times washed with 0-9% sodium
C. L. CAZZULLO.
DIVORCE REFORM
Sir,- was pleased to read the account in your parliamentary columns (Feb. 17, p. 365) of the second reading of Mr. William Wilson’s important but controversial Bill on divorce reform. However, your report’s reference to insanity cases requires some clarification. Incurable insanity first became a ground for divorce proceedings as long ago as 1937. The requirements are set out in the Matrimonial Causes Act. These are that the patient is incurably of unsound mind and has been continuously under care and treatment for a period of five years. Various difficulties have arisen as to the interpretation and the practical application of these criteria. Firstly, as has been pointed out,! there is a divergence of judicial opinion on whether a person suffering from subnormality can be said to be a person of " unsound mind ". Secondly, it has become very difficult in the light of the great advances in psychiatry to prove that insanity is incurable. Finally, the Law as it stands is objectionable in that it equates the misfortune of insanity with a matrimonial offence. The new Bill overcomes all these points by not specifically mentioning insanity at all. The test is whether the marriage has broken down irretrievably, and this is to be inferred not so much from insanity as from the respondent’s unreasonable behaviour, or from the fact that the parties have lived apart for five years. As Mr. Wilson said2 in introducing his Bill, the question of insanity is covered more effectively than under the present Law, and in a more realistic and humane fashion. I feel that at least in this respect the Bill deserves the full support of the medical profession. Monyhull Hospital, Birmingham 30.
A. I. ROITH.
1. Roith, A. I. Lancet, 1967, i, 49. ibid. p. 154. 2. House of Commons Hansard, Feb. 9, 1968, col. 814.
Electrophoresis of pyruvate kinase, photographed in ultraviolet light. A. Increased enzyme activity. B. Partial enzyme C. Usual enzyme.
deficiency.
chloride
solution) 1-00 ml.; digitonin (saturated solution), ml.; distilled water, 0-67 ml.; 0-2 Methylene diamine tetra-acetic acid (E.D.T.A.), 0-02 ml.; and (2 x 10 w4 M) 2-mercaptoethanol, 0-01 ml. The mixture is rapidly frozen and thawed twice for complete haemolysis, then centrifuged for 20 minutes at 40,000 g. The haemoglobin concentration in the supernatant is about 12’0 g. per 100 ml. Cellogel strips (0-25 x 40 x 170 mm.) 0-30
phosphate buffer (pH 6,2) containing (2 10-6M)2-mercaptoethanol and (4 x 10-4 M) E.D.T.A. This incubation is repeated twice for 10 minutes each are
incubated for 1 hour in 3
x
10-2 M
x
time with fresh buffer. The samples (about 3 litres) are applied on the porous surface of the cellogel strips in the form of thin streaks, 5 mm. long at the origin, 60 mm. from the cathodic end, using a 1.
Dioguardi, N., Agostini, A., Fiorelli, G., Lomanto, B. J. Lab. clin. Med.
1963, 61, 713. Rattazzi, M. C., Bernini, L. F., Fiorelli, G., Mannucci, P. M. Nature, Lond. 1967, 213, 79. 3. Blume, K. G., Rüdiger, H. W., Löhr, G. W. Biochim. biophys. Acta (in the press). 4. Loos, J. A., Prins, H. K. in International Symposium on Hereditary Enzymatic Deficiencies of Red Blood Cells, Duarte, California, Feb. 16, 1967 (in the press). 5. Valentine, W. N., Tanaka, K. R., Miwa, S. Trans. Ass. Am. Physns, 1961, 74, 100. Waller, H. D., Löhr, G. W. Proceedings of the 9th Congress of the International Society of Hæmatology, vol. I, p. 257. Mexico City, 1962. 2.
530
micropipette. Within 1 minute the samples are absorbed on the gel. Linen wicks with’Cellophane’ covers are used. The cathodic chamber is filled with phosphate buffer (4-5 x 10-2 M, pH 62). The anodic chamber contains phosphate buffer (1-5x 10-2 M, pH 6-2). The concentrations of 2-mercaptoethanol and E.D.T.A. correspond to the amounts mentioned above. The run usually takes 100 minutes at 4 mA per strip, at about 1000 V and -4°C. The staining solution consists of 5-0 x 10-2 M trishydroxymethylaminomethane buffer (pH 7.5), 4-0 x 10-2 M potassium chloride, 2-5 x 10-2 M magnesium chloride, 7-0 x 10-3 M nicotinamide adenosine dinucleotide (reduced form) N.A.D.2H, 6-0 x 10-3 M phosphoenolpyruvate, and 0-02 ml. crystallised lactic dehydrogenase (0-1 mg.). A filter paper (40 x 170 mm.) is soaked with 1 ml. of this solution, and applied to the porous surface of the strip. After incubation
manifestations of
for 10 minutes at 37°C the result can be observed in ultraviolet light. In zones of pyruvate kinase the bright fluorescence of N.A.D.2H is extinguished. At these places no extinction of fluorescence will appear if no phosphoenolpyruvate is present in the staining solution. K. G. BLUME G. W. LÖHR W. RÜDIGER H. Medical Policlinic, A. SCHALHORN. 355 Marburg/Lahn, Germany.
found in the common bileduct. Only 11 cases have previously been reported,7-9 and, of these, only 3 78 could possibly have been due to ingestion. I report here a case of a seed of the jujube fruit in the common bileduct, seen in this hospital. The patient, the forty-year-old wife of a farmer, was admitted to the surgical ward after having had abdominal pain for five days. She had been first seen at the outpatient clinic on Feb. 12, 1964, three years before, with pain in the right iliac fossa, and had been treated for chronic appendicitis, with a single course of antibiotic and analgesic. She had been well for the next three years. Twenty days before this admission, she had been admitted to the medical ward for fever of unknown cause. On the third day of that admission, she had dull, aching, right-sided abdominal pain, with localised moderate tenderness in the right hypochondrium. 5 hookworm ova were found in a slide of the second stool sample, and hookworm duodenitis was diagnosed, for which she was given chloramphenicol, 1-5 g. daily, and supportive treatment. A stool sample, collected two days after a single dose of 2-5 g. of bephenium hydroxynaphthoate (’ Alcopar ’), was negative. She was discharged on the twelfth day, after having been symptom-free for five days. The patient was readmitted on the eighth day after her discharge. Five days before she had had a sharp sudden pain in the right hypochondrium, lasting a few minutes. The pain had returned in bouts which became more frequent and lasted longer, and had developed into a constant aching on the last two days. She had anorexia, but no nausea, vomiting, or referred pain. The patient was short and slim, looked about ten years older than her age, and was slightly dehydrated and jaundiced. A point of maximal tenderness was located in the right hypochondrium; muscular rigidity was moderate, and a cystic mass, the size of a lemon, was palpable underneath. Murphy’s sign was obviously positive. Blood-count was
DYSAUTONOMIA AND AMINOACIDURIA SiR,—The increased homovanillic-acid/vannilylmandelicacid ratio reported1 in the urine of children with familial dysautonomia suggests that this disease must be considered among inborn errors of metabolism. We have reported2 on two children, 4 and 13 years old, with the typical clinical and laboratory features of dysautonomia.3 One of these patients, studied by Technicon aminoacidAutoanalyser’, showed a normal level of plasma-aminoacids; the urinary excretion of aminoacids and aminoacid products is shown in the accompanying table. A similar, but not so striking, pattern of aminoaciduria was seen in the patient’s mother, who had no 1.
Smith,
A. A.,
Taylor, T., Wortis,
S. B. New
Engl. J. Med. 1963, 268,
705.
Rosano, M., Ammaniti, M., Lapi, A. Infanz. anorm. 1966, 72, 976. Riley, C. M., Moore, R. H. Pediatrics, Springfield, 1966, 37, 435. 4. Spackman, D. H., Stein, W. H., Moore, S. Analyt. Chem. 1958, 30, 2. 3.
1190. 5.
Stein, W. H. J. biol. Chem. 1943, 150,
URINARY EXCRETION
251.
OF AMINOACIDS AND AMINOACID PRODUCTS IN PATIENT AND 2 CONTROL SOURCES
dysautonomia, but not in other relatives investigated. These findings pose the question whether the aminoaciduria is primary or secondary in dysautonomia.There have reports of defective aminoacid metabolism in any other with this disease. The aminoaciduria in our patient, also shown by the mother, therefore seems to be a genetically transmitted enzymatic error which interferes with tubular been
no
patients
reabsorption. Institute of Pædiatric Neuropsychiatry, Rome University.
M. ROSANO M. AMMANITI.
JUJUBE IN COMMON BILEDUCT SIR,-Foreign bodies, particularly non-parasitic,
are
rarely
11,000 leucocytes per c.mm. (78% neutrophils, 2% eosinophils,
20% lymphocytes). A stool sample showed two hookworm ova in a slide. Cholecystography showed no dye excretion, even in the film taken at the end of two and a half hours. The sulphobromophthalein-sodium test showed 46% dye retention. Cholecystitis and obstructive jaundice were diagnosed. At operation, on the tenth day, the gallbladder was thickened and loosely adherent to the first part of duodenum. There was moderate fibrous adhesion around the right subhepatic space. The common bileduct was 1/3 in. (0-8 cm.) in diameter and thickened, and complete palpation was not possible through the foramen of Winslow. The appendix was normal and without evidence of recent inflammation. The gallbladder was removed without difficulty; the common duct was explored through a choledochotomy opening placed distal to the level of the cystic duct opening, and an oval body, with an attached stalk pointing downward, was extracted from that site. The patient recovered well and was discharged on the seventeenth day after operation. Apart from a slightly painful, hypertrophic scar, she
The
was
well when
foreign body 6. 7. 8. 9.
four months later. encrusted with bile precipitate, and
seen
was
Efron, M. L. New Engl. J. Med. 1965, 272, 1058, Bowen, F. H. Archs Surg., Chicago, 1941, 43, 458. Byrne, J. J. ibid. 1955, 71, 91. Connel, J. L. Br. J. Surg. 1954, 42, 418.
1107.
1.—PERCENTAGE
COMPOSITION
OF
PLATELET CHOLESTEROL, IN NORMAL SUBJECTS
TRIGLYCERIDES, AND TOTAL PHOSPHOLIPIDS AND MULTIPLE-SCLEROSIS PATIENTS
Each
figure represents
mean
of 5
experiments ::1=
s.E.
TABLE II.-PERCENTAGE COMPOSITION OF SINGLE PLATELET-PHOSPHOLIPID FRACTIONS OF NORMAL SUBJECTS AND MULTIPLE-SCLEROSIS PATIENTS
Numbers of
experiments
are
shown in
parentheses.
The results indicate that the differences in metabolic response and sensitivity to A.D.P. observed in platelets of M.S. patients are not due to changes of the major neutral lipids and phospholipids. Plasma alterations, as described by Dr. Bolton and his colleagues, may therefore be the important factor in platelet behaviour in patients with M.S., as indirectly confirmed by our results. However, the possibility that alterations in long-chain fatty-acid composition of phospholipids present in plateletmembranes may also play a relevant role should be considered. Investigations are being carried out in our laboratory along both these lines. V. M. ANDREOLI. Department of Pharmacology, Multiple Sclerosis Centre, University of Milan, Italy.
PYRUVATE KINASE IN HUMAN ERYTHROCYTES
SIR,-We report here a technique for the separation of pyruvate-kinase isozymes from human erythrocytes by electrophoresis on cellulose acetate in a gel form (’ Cellogel ’, Chemetron, Milan, Italy); cellogel has been used for investigating lactic dehydrogenase isozymes,1 glucose-6-phosphate dehydrogenase,2and glutathione-reductase isozymes.3 By high voltage electrophoresis at pH 62 two distinct isozymes can be observed, one band (PK I) moving towards the anode, the other (PK II) towards the cathode (see accompanying figure). In patients with increased enzyme activity 4 (A, 92 units per 1011 erythrocytes), which are associated with augmented adenosine-triphosphate-values in the erythrocytes, the PK I spot is larger than PK I of the usual enzyme (C, 40 units per 1011 erythrocytes). In patients with partial deficiency of pyruvate kinase (B, 16 units per 1011 erythrocytes), who clinically show a hamiolytic, non-spherocytic anxmia,5 the spot of PK I is smaller and moves more slowly from the origin. The mobility of the PK II isozymes seems to be similar in all three cases. In white blood-cells and platelets there is only one isozyme, which moves very slowly from the origin towards the cathode. For preparation of hoemolysates, the following mixture is used: erythrocytes (three times washed with 0-9% sodium
C. L. CAZZULLO.
DIVORCE REFORM
Sir,- was pleased to read the account in your parliamentary columns (Feb. 17, p. 365) of the second reading of Mr. William Wilson’s important but controversial Bill on divorce reform. However, your report’s reference to insanity cases requires some clarification. Incurable insanity first became a ground for divorce proceedings as long ago as 1937. The requirements are set out in the Matrimonial Causes Act. These are that the patient is incurably of unsound mind and has been continuously under care and treatment for a period of five years. Various difficulties have arisen as to the interpretation and the practical application of these criteria. Firstly, as has been pointed out,! there is a divergence of judicial opinion on whether a person suffering from subnormality can be said to be a person of " unsound mind ". Secondly, it has become very difficult in the light of the great advances in psychiatry to prove that insanity is incurable. Finally, the Law as it stands is objectionable in that it equates the misfortune of insanity with a matrimonial offence. The new Bill overcomes all these points by not specifically mentioning insanity at all. The test is whether the marriage has broken down irretrievably, and this is to be inferred not so much from insanity as from the respondent’s unreasonable behaviour, or from the fact that the parties have lived apart for five years. As Mr. Wilson said2 in introducing his Bill, the question of insanity is covered more effectively than under the present Law, and in a more realistic and humane fashion. I feel that at least in this respect the Bill deserves the full support of the medical profession. Monyhull Hospital, Birmingham 30.
A. I. ROITH.
1. Roith, A. I. Lancet, 1967, i, 49. ibid. p. 154. 2. House of Commons Hansard, Feb. 9, 1968, col. 814.
Electrophoresis of pyruvate kinase, photographed in ultraviolet light. A. Increased enzyme activity. B. Partial enzyme C. Usual enzyme.
deficiency.
chloride
solution) 1-00 ml.; digitonin (saturated solution), ml.; distilled water, 0-67 ml.; 0-2 Methylene diamine tetra-acetic acid (E.D.T.A.), 0-02 ml.; and (2 x 10 w4 M) 2-mercaptoethanol, 0-01 ml. The mixture is rapidly frozen and thawed twice for complete haemolysis, then centrifuged for 20 minutes at 40,000 g. The haemoglobin concentration in the supernatant is about 12’0 g. per 100 ml. Cellogel strips (0-25 x 40 x 170 mm.) 0-30
phosphate buffer (pH 6,2) containing (2 10-6M)2-mercaptoethanol and (4 x 10-4 M) E.D.T.A. This incubation is repeated twice for 10 minutes each are
incubated for 1 hour in 3
x
10-2 M
x
time with fresh buffer. The samples (about 3 litres) are applied on the porous surface of the cellogel strips in the form of thin streaks, 5 mm. long at the origin, 60 mm. from the cathodic end, using a 1.
Dioguardi, N., Agostini, A., Fiorelli, G., Lomanto, B. J. Lab. clin. Med.
1963, 61, 713. Rattazzi, M. C., Bernini, L. F., Fiorelli, G., Mannucci, P. M. Nature, Lond. 1967, 213, 79. 3. Blume, K. G., Rüdiger, H. W., Löhr, G. W. Biochim. biophys. Acta (in the press). 4. Loos, J. A., Prins, H. K. in International Symposium on Hereditary Enzymatic Deficiencies of Red Blood Cells, Duarte, California, Feb. 16, 1967 (in the press). 5. Valentine, W. N., Tanaka, K. R., Miwa, S. Trans. Ass. Am. Physns, 1961, 74, 100. Waller, H. D., Löhr, G. W. Proceedings of the 9th Congress of the International Society of Hæmatology, vol. I, p. 257. Mexico City, 1962. 2.
530
micropipette. Within 1 minute the samples are absorbed on the gel. Linen wicks with’Cellophane’ covers are used. The cathodic chamber is filled with phosphate buffer (4-5 x 10-2 M, pH 62). The anodic chamber contains phosphate buffer (1-5x 10-2 M, pH 6-2). The concentrations of 2-mercaptoethanol and E.D.T.A. correspond to the amounts mentioned above. The run usually takes 100 minutes at 4 mA per strip, at about 1000 V and -4°C. The staining solution consists of 5-0 x 10-2 M trishydroxymethylaminomethane buffer (pH 7.5), 4-0 x 10-2 M potassium chloride, 2-5 x 10-2 M magnesium chloride, 7-0 x 10-3 M nicotinamide adenosine dinucleotide (reduced form) N.A.D.2H, 6-0 x 10-3 M phosphoenolpyruvate, and 0-02 ml. crystallised lactic dehydrogenase (0-1 mg.). A filter paper (40 x 170 mm.) is soaked with 1 ml. of this solution, and applied to the porous surface of the strip. After incubation
manifestations of
for 10 minutes at 37°C the result can be observed in ultraviolet light. In zones of pyruvate kinase the bright fluorescence of N.A.D.2H is extinguished. At these places no extinction of fluorescence will appear if no phosphoenolpyruvate is present in the staining solution. K. G. BLUME G. W. LÖHR W. RÜDIGER H. Medical Policlinic, A. SCHALHORN. 355 Marburg/Lahn, Germany.
found in the common bileduct. Only 11 cases have previously been reported,7-9 and, of these, only 3 78 could possibly have been due to ingestion. I report here a case of a seed of the jujube fruit in the common bileduct, seen in this hospital. The patient, the forty-year-old wife of a farmer, was admitted to the surgical ward after having had abdominal pain for five days. She had been first seen at the outpatient clinic on Feb. 12, 1964, three years before, with pain in the right iliac fossa, and had been treated for chronic appendicitis, with a single course of antibiotic and analgesic. She had been well for the next three years. Twenty days before this admission, she had been admitted to the medical ward for fever of unknown cause. On the third day of that admission, she had dull, aching, right-sided abdominal pain, with localised moderate tenderness in the right hypochondrium. 5 hookworm ova were found in a slide of the second stool sample, and hookworm duodenitis was diagnosed, for which she was given chloramphenicol, 1-5 g. daily, and supportive treatment. A stool sample, collected two days after a single dose of 2-5 g. of bephenium hydroxynaphthoate (’ Alcopar ’), was negative. She was discharged on the twelfth day, after having been symptom-free for five days. The patient was readmitted on the eighth day after her discharge. Five days before she had had a sharp sudden pain in the right hypochondrium, lasting a few minutes. The pain had returned in bouts which became more frequent and lasted longer, and had developed into a constant aching on the last two days. She had anorexia, but no nausea, vomiting, or referred pain. The patient was short and slim, looked about ten years older than her age, and was slightly dehydrated and jaundiced. A point of maximal tenderness was located in the right hypochondrium; muscular rigidity was moderate, and a cystic mass, the size of a lemon, was palpable underneath. Murphy’s sign was obviously positive. Blood-count was
DYSAUTONOMIA AND AMINOACIDURIA SiR,—The increased homovanillic-acid/vannilylmandelicacid ratio reported1 in the urine of children with familial dysautonomia suggests that this disease must be considered among inborn errors of metabolism. We have reported2 on two children, 4 and 13 years old, with the typical clinical and laboratory features of dysautonomia.3 One of these patients, studied by Technicon aminoacidAutoanalyser’, showed a normal level of plasma-aminoacids; the urinary excretion of aminoacids and aminoacid products is shown in the accompanying table. A similar, but not so striking, pattern of aminoaciduria was seen in the patient’s mother, who had no 1.
Smith,
A. A.,
Taylor, T., Wortis,
S. B. New
Engl. J. Med. 1963, 268,
705.
Rosano, M., Ammaniti, M., Lapi, A. Infanz. anorm. 1966, 72, 976. Riley, C. M., Moore, R. H. Pediatrics, Springfield, 1966, 37, 435. 4. Spackman, D. H., Stein, W. H., Moore, S. Analyt. Chem. 1958, 30, 2. 3.
1190. 5.
Stein, W. H. J. biol. Chem. 1943, 150,
URINARY EXCRETION
251.
OF AMINOACIDS AND AMINOACID PRODUCTS IN PATIENT AND 2 CONTROL SOURCES
dysautonomia, but not in other relatives investigated. These findings pose the question whether the aminoaciduria is primary or secondary in dysautonomia.There have reports of defective aminoacid metabolism in any other with this disease. The aminoaciduria in our patient, also shown by the mother, therefore seems to be a genetically transmitted enzymatic error which interferes with tubular been
no
patients
reabsorption. Institute of Pædiatric Neuropsychiatry, Rome University.
M. ROSANO M. AMMANITI.
JUJUBE IN COMMON BILEDUCT SIR,-Foreign bodies, particularly non-parasitic,
are
rarely
11,000 leucocytes per c.mm. (78% neutrophils, 2% eosinophils,
20% lymphocytes). A stool sample showed two hookworm ova in a slide. Cholecystography showed no dye excretion, even in the film taken at the end of two and a half hours. The sulphobromophthalein-sodium test showed 46% dye retention. Cholecystitis and obstructive jaundice were diagnosed. At operation, on the tenth day, the gallbladder was thickened and loosely adherent to the first part of duodenum. There was moderate fibrous adhesion around the right subhepatic space. The common bileduct was 1/3 in. (0-8 cm.) in diameter and thickened, and complete palpation was not possible through the foramen of Winslow. The appendix was normal and without evidence of recent inflammation. The gallbladder was removed without difficulty; the common duct was explored through a choledochotomy opening placed distal to the level of the cystic duct opening, and an oval body, with an attached stalk pointing downward, was extracted from that site. The patient recovered well and was discharged on the seventeenth day after operation. Apart from a slightly painful, hypertrophic scar, she
The
was
well when
foreign body 6. 7. 8. 9.
four months later. encrusted with bile precipitate, and
seen
was
Efron, M. L. New Engl. J. Med. 1965, 272, 1058, Bowen, F. H. Archs Surg., Chicago, 1941, 43, 458. Byrne, J. J. ibid. 1955, 71, 91. Connel, J. L. Br. J. Surg. 1954, 42, 418.
1107.