- Email: [email protected]
INDUCED METABOLIC ALKALOSIS IN HEPATIC ENCEPHALOPATHY
1364 the patient’s urine. The paper immediately lost colour. The fact that this reversal of the colour reaction occurred with the minimal concentration of the alkaptonuric urine which prevented the positive colour reaction from developing in the presence of glucose suggests that reduction of the chromogen is at least as important as direct inhibition of either of the enzymes involved in the reaction. The alkaptonuric urine containing glucose was then tested by dipping only the end of the test-paper into the urine, rather than completely immersing it as we had done previously. When this was done, a thin line of colour indicating a positive reaction was noted at the diffusion front on the top of the tape. Gifford and Bergerman also noted this type of reaction with several drugs which inhibit the glucose-oxidase tape reaction.2 Presumably all these compounds have a lower Rf value than glucose in this mini-ascending-chromatography system, so that at the front no inhibitor is present and a positive reaction can develop. This technique has allowed our patient with alkaptonuria to solve the dilemma of detecting glycosuria. WILLIAM N. KELLEY Division of Metabolic IRVING H. FOX and Genetic Diseases, M. FELDMAN JEROME Duke University Medical Center, JAMES B. WYNGAARDEN. Durham, North Carolina.
SUGAR INTAKE AND CORONARY HEART-DISEASE SIR,-With regard to Dr. Walker’s letter (Nov. 15, p. 1071), may I submit that it is fallacious to compare the incidence of coronary disease in Chandrigarh, North India, with that in Tecumseh, U.S.A. ? He is comparing two different races. In a joint work3 there are several pages showing how the genetic susceptibility to new factors in the environment varies greatly between different races. This is strikingly seen, for example, between the Indians and Africans in Natal, in the case of diabetes-with which coronary thrombosis is closely associated. I contend that a comparison between the Indians in Chandrigarh and those in Natal would show a very different result. T. L. CLEAVE. Fareham, Hants.
NON-PULMONARY TUBERCULOSIS
SIR,-At two meetings on tuberculosis which I have recently attended, none of the speakers mentioned nonpulmonary tuberculosis. Yet in some areas it is an important and growing problem. A boy of 10 came into my surgery with a large swelling of the upper arm and elbow region. The differential diagnosis included osteomyelitis, bone abscess, sarcoma, and arthritis. On referral to a teaching-hospital, the diagnosis of tuberculosis of bone and joint was quickly made. The boy was a Pakistani, and, in retrospect, the possibility of tubercle should have been first in my mind. After this incident, I approached other general practitioners ; several had encountered cases of non-pulmonary tuberculosis in the immigrant population. I have also heard, from hospital sources, of cases of psoas abscess and Pott’s disease (the names sound like echoes of battles long ago) where the diagnosis changed half-a-dozen times before the disease
was
identified.
Perhaps it is too easily assumed that the problem of nonpulmonary tuberculosis is negligible nowadays. Nonpulmonary infection is nowadays seldom caused by the bovine bacillus; it is nearly 100% human type. The occurrence of an index case therefore should be a pointer to 2.
Gifford, H., Bergerman, J. J. Am. med. Ass. 1961, 178, 423. Campbell, G. D., Painter, N. S. Diabetes, Coronary Thrombosis, and the Saccharine Diseases. Bristol, 1969.
3. Cleave, T. L.,
human source capable of spreading infection further. The community health team (family doctor, health visitor, district nurse) has an important role in spotting such cases. With the arrival in some areas of a more susceptible population, the problem appears to be a growing one. It is also an expensive one, not only in terms of cost, but also in length of treatment and other demands on medical a
and social
care.
J. L. BURN.
Salford.
INDUCED METABOLIC ALKALOSIS IN HEPATIC ENCEPHALOPATHY
SIR,-The observations of Dr. James and his colleagues (Nov. 22, p. 1106) on the beneficial effect of infused sodium bicarbonate in hepatic encephalopathy prompt us to draw your attention to some experimental data of our own on brain slices 1.2 which may provide further support for their work. The oxygen uptake of potassium-enriched rat-brain slices was studied in vitro in circumstances mimicking uraemic comaand hepatic coma.2 When a fall of pH of 0.2 units was induced in the incubating medium, the mean oxygen uptake fell1 (see accompanying table). When the pH was increased, the oxygen uptake increased. EFFECT OF
pH
CHANGES ON OXYGEN UPTAKE OF BRAIN SLICES
In each of these groups of experiments the test observations did not differ significantly from the controls, nor did the two test values, when corrected for the difference between the controls, differ significantly (0-1 >P> 0.05). The trend of these results attracted our attention, nevertheless, for two reasons. Firstly, an enhanced oxygen uptake was a rare event in this series of experiments, occuring only with increasing pH and with potassium enrichment of the medium. Secondly, the results are in marked contrast to those with kidney and with liver slices, which did not show such a trend. With kidney slices, when the pH was lowered by 0-5 units, the oxygen uptake was 96-5% and, when the pH was raised by 0-4 units, the uptake was 98-0%. With liver slices the comparable values were 72-0% and 74-5%. It was found, infurther experiments, that the simultaneous addition to the incubating medium of several of the metabolites which accumulate in hepatic coma would greatly inhibit the oxygen uptake of brain slices; the oxygen uptake during the third hour was 30% (controls 77’5%). This inhibition could be substantially and significantly overcome (P<0’01) by raising the pH of the mixture of metabolites; when the pH was raised by 1.5 units the oxygen
uptake
was as
high
as
77-3% (controls 90%).
Our conclusion was, " the rise (of pH) protects the slices from the slow inhibition that occurs with time and from the inhibition that can be induced by a mixture of metabolites ... These observations raise the interesting possibility that the alkalosis of hepatic coma may be beneficial to cerebral metabolism". It is gratifying to find that this evidence and that of Dr. James and his colleagues should fit so well together, despite the different experimen1. 2.
Lascelles, P. T., Taylor, W. H. Clin. Sci. 1966, 31, 403. Lascelles, P. T., Taylor, W. H. ibid. 1968, 35, 63.
1365
tal circumstances in which the
two sets
Departments of Chemical Pathology, National Hospital, Queen Square, London W.C.1, and United Liverpool Hospitals, Liverpool, L3 5RT.
P. T. LASCELLES W. H. TAYLOR.
POLYPLOIDY SIR,-Polyploidy of human lymphocytes has been observed in malignant disease,1,2 after irradiation, and following administration of various drugs.3,4 Abnormalities of E-group chromosomes and increases in chromosomal breaks, on the other hand, are associated with disturbances in production of immunoglobulins.5-8 We have observed chromosomal polyploidy in a patient with immunoglobulin
deficiency. The patient was a 29-year-old farm labourer who had a history of frequent pulmonary infection since the age of 5, when he had whooping-cough. In March, 1968, he was admitted to hospital with pneumonia. He was of short stature (height 150 cm.), his i.Q. was 63, and he had had an operation on his right hand for polydactyly. The peripheralleucocyte count was within normal limits (lymphocytes did not exceed 15%). In bone-marrow smears plasma-cells were totally absent. Serum-immunoglobulin assay showed that IgA and IgM were absent, and IgG were considerably diminished. Various bacterial antibody titres were diminished or undetectable. Chromosome studies were done on 48-hour cultures of peripheral leucocytes stimulated by phytohaemagglutinin, and directly from sternal bone-marrow cells obtained by needle aspiration. Among the 33 photographed mitoses there were 8 polyploid cells, with chromosome numbers ranging from 63 to 93. The diploid cells showed a normal male (46,XY) karyotype. There were chromosomal breaks in 9 of the diploid cells, and in 3 of these chromosome number 2 was dicentric. E-group chromosomes were normal. Among the 31 karyotyped bone-marrow mitoses there were 4 polyploid cells. The diploid cells in the bonemarrow cells had normal karyotypes, with the exception of 1 with a supernumerary E-group chromosome. Further observations in other patients will be needed in order to ascertain whether this conspicuous polyploidy was related to the immunoglobulin deficiency. There is also the that polyploidy was an early sign of malignant disease. This is common in patients with immunoglobulin deficiencies,9 but so far there are no signs of lymphoma in
possibility
patient.
A detailed
description of this case will be published elsewhere. Department of Pathology, Robert Károly Hospital, M. SELLYEI. Budapest XIII. Department of Medicine, National Institute of Rheumatology, GABRIELLA KISS. Budapest III. State Human " Serum Institute, R. BACKHAUSZ. Budapest X. "
1. 2. 3.
toxicity 476) are interesting and thought-provoking. You conclude by saying that the use of chloramphenicol should be restricted to patients with typhoid fever or other gram-negative infections where no suitable alternative antibiotic is available; this statement needs some qualification. Typhoid fever is primarily a disease of developing (Aug. 30,
IMMUNOGLOBULIN DEFICIENCY AND
our
DRUGS FOR TYPHOID FEVER SIR,-Your remarks about chloramphenicol
of data have been
obtained.
Lancet, 1964, ii, 511. Bauke, J., Schöffling, K. Cancer, Philad. 1968, 22, 686. Friedman, B. I., Saenger, E. L., Kreindler, M. S. Lancet, 1964, ii, 494. 4. Nasjleti, C. E., Spencer, H. H. Cancer, Philad. 1967, 20, 31. 5. Haddad, Z. H., Allen, R. F., Towner, J. W., Wilson, M. G. Lancet, 1969, i, 678. 6. Hecht, F. ibid. p. 100. 7. Jacobs, J. C., de Capoa, A., McGilvray, E., Morse, J. H., Schullinger, J. N., Blanc, W. A., Heird, W. C., Miller, O. J., Rossen, R. D., Walzer, R. A. ibid. 1968, i, 499. 8. Hecht, F., Koler, R. D., Rigas, D. A., Dahnke, G. S., Case, M. P., Tisdale, V., Miller, R. W. ibid. 1966, ii, 1193. 9. ibid. 1969, i, 163.
p.
countries. The incidence of enteric fever in West Pakistan has been estimated to be about 1 %. On this basis, half a million cases probably occur in West Pakistan every year. Only six thousand of these patients are admitted to Government hospitals.1 The majority are treated outside the hospital by general practitioners. In general practice, as in the hospitals, treatment is normally instituted immediately upon clinical diagnosis of typhoid fever. Confirmation by serological tests is not awaited in most cases.2 Serological and blood-culture facilities are not widely available in the countries where typhoid is most prevalent, and where available they are expensive and time-consuming. Under these circumstances, a large number of patients, supposedly having typhoid fever, are exposed to the hazards of
chloramphenicol. Furazolidone, a nitrofuran derivative, has recently been extensively tried in the treatment of typhoid fever in India,3-1o Pakistan,11,12 and elsewhere.13,14 The results have been
encouraging. Furazolidone has also been used for disorders of the gastrointestinal tract for more than 8 years, and no serious toxic reactions have been reported. Under these circumstances, I feel that furazolidone should be tried first in all cases of typhoid fever. I think you will agree that its use in the clinically diagnosed cases of typhoid fever will serve to protect patients from unnecessary exposure to In my opinion, chloramphenicol should patients with typhoid fever who require parenteral therapy or who fail to respond to furazolidone. No cross-resistance has been reported between the two drugs.
chloramphenicol.
be reserved for
Jinnah Postgraduate Medical Centre, Karachi, Pakistan.
ANWAR MASOOD.
ŒSOPHAGEAL CANCER IN AFRICA
SIR,-It is possible that the apparent great increase in cancer of the oesophagus in parts of Africa, to which you refer in your annotation (Nov. 29, p. 1178), is due partly to increased longevity. Palmar and plantar keratosis, which I suspect is common in Africa, is one condition associated with cancer of the oesophagus in late middle life. (It is a long time since I worked in West and South Africa, and I did not know about this familial disease syndrome at the time, but I remember wondering why some Africans had thickened palms though they were not employed in hard manual labour.) Even in Britain there are no good records of the diseases of past generations. I believe that when such records become available, certain forms of cancer will be found to be associated with developmental anomalies. In relation to palmar keratosis in Africans, it would be important to Awan, A. H. Pakist. J. med. Res. 1969, 8, 91. Hameedi, A. A. J. Pakist. med. Ass. 1964, 14, 745. Chakraborty, G. Indian J. Pœdiat. 1961, 28, 164. Chakraborty, G. J. Indian med. Ass. 1958, 41, 10. Damany, S. J., Bilgi, C. L. J. Ass. Phys. India, 1968, 16, 669. Jaffari, S. M. H. Punjab med. J. 1966, 15, 423. Kamat, S. A. J. Am. med. Ass. 1968, 206, 2745. Luhar, P. M. Clin. Trials J. 1967, 4, 816. Mehta, M. R. Indian Practitioner, 1967, 20, 327. Solanki, S. V., Kabrawala, V. N. Saurashtra Univ. J. 1968, I, 155. Khan, N. M. Medicus, 1969, 38, 258. Razzaq, A., Hayee, A. Pakist. J. med. Res. (in the press). Musgrave, M. E., Brownlow, W. J. Cited by Jaffari, S. M. H. Punjab med. J. 1966, 15, 423. 14. Paul, M. R. Antibiotics Chemother. 1960, 10, 231. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
SUGAR INTAKE AND CORONARY HEART-DISEASE SIR,-With regard to Dr. Walker’s letter (Nov. 15, p. 1071), may I submit that it is fallacious to compare the incidence of coronary disease in Chandrigarh, North India, with that in Tecumseh, U.S.A. ? He is comparing two different races. In a joint work3 there are several pages showing how the genetic susceptibility to new factors in the environment varies greatly between different races. This is strikingly seen, for example, between the Indians and Africans in Natal, in the case of diabetes-with which coronary thrombosis is closely associated. I contend that a comparison between the Indians in Chandrigarh and those in Natal would show a very different result. T. L. CLEAVE. Fareham, Hants.
NON-PULMONARY TUBERCULOSIS
SIR,-At two meetings on tuberculosis which I have recently attended, none of the speakers mentioned nonpulmonary tuberculosis. Yet in some areas it is an important and growing problem. A boy of 10 came into my surgery with a large swelling of the upper arm and elbow region. The differential diagnosis included osteomyelitis, bone abscess, sarcoma, and arthritis. On referral to a teaching-hospital, the diagnosis of tuberculosis of bone and joint was quickly made. The boy was a Pakistani, and, in retrospect, the possibility of tubercle should have been first in my mind. After this incident, I approached other general practitioners ; several had encountered cases of non-pulmonary tuberculosis in the immigrant population. I have also heard, from hospital sources, of cases of psoas abscess and Pott’s disease (the names sound like echoes of battles long ago) where the diagnosis changed half-a-dozen times before the disease
was
identified.
Perhaps it is too easily assumed that the problem of nonpulmonary tuberculosis is negligible nowadays. Nonpulmonary infection is nowadays seldom caused by the bovine bacillus; it is nearly 100% human type. The occurrence of an index case therefore should be a pointer to 2.
Gifford, H., Bergerman, J. J. Am. med. Ass. 1961, 178, 423. Campbell, G. D., Painter, N. S. Diabetes, Coronary Thrombosis, and the Saccharine Diseases. Bristol, 1969.
3. Cleave, T. L.,
human source capable of spreading infection further. The community health team (family doctor, health visitor, district nurse) has an important role in spotting such cases. With the arrival in some areas of a more susceptible population, the problem appears to be a growing one. It is also an expensive one, not only in terms of cost, but also in length of treatment and other demands on medical a
and social
care.
J. L. BURN.
Salford.
INDUCED METABOLIC ALKALOSIS IN HEPATIC ENCEPHALOPATHY
SIR,-The observations of Dr. James and his colleagues (Nov. 22, p. 1106) on the beneficial effect of infused sodium bicarbonate in hepatic encephalopathy prompt us to draw your attention to some experimental data of our own on brain slices 1.2 which may provide further support for their work. The oxygen uptake of potassium-enriched rat-brain slices was studied in vitro in circumstances mimicking uraemic comaand hepatic coma.2 When a fall of pH of 0.2 units was induced in the incubating medium, the mean oxygen uptake fell1 (see accompanying table). When the pH was increased, the oxygen uptake increased. EFFECT OF
pH
CHANGES ON OXYGEN UPTAKE OF BRAIN SLICES
In each of these groups of experiments the test observations did not differ significantly from the controls, nor did the two test values, when corrected for the difference between the controls, differ significantly (0-1 >P> 0.05). The trend of these results attracted our attention, nevertheless, for two reasons. Firstly, an enhanced oxygen uptake was a rare event in this series of experiments, occuring only with increasing pH and with potassium enrichment of the medium. Secondly, the results are in marked contrast to those with kidney and with liver slices, which did not show such a trend. With kidney slices, when the pH was lowered by 0-5 units, the oxygen uptake was 96-5% and, when the pH was raised by 0-4 units, the uptake was 98-0%. With liver slices the comparable values were 72-0% and 74-5%. It was found, infurther experiments, that the simultaneous addition to the incubating medium of several of the metabolites which accumulate in hepatic coma would greatly inhibit the oxygen uptake of brain slices; the oxygen uptake during the third hour was 30% (controls 77’5%). This inhibition could be substantially and significantly overcome (P<0’01) by raising the pH of the mixture of metabolites; when the pH was raised by 1.5 units the oxygen
uptake
was as
high
as
77-3% (controls 90%).
Our conclusion was, " the rise (of pH) protects the slices from the slow inhibition that occurs with time and from the inhibition that can be induced by a mixture of metabolites ... These observations raise the interesting possibility that the alkalosis of hepatic coma may be beneficial to cerebral metabolism". It is gratifying to find that this evidence and that of Dr. James and his colleagues should fit so well together, despite the different experimen1. 2.
Lascelles, P. T., Taylor, W. H. Clin. Sci. 1966, 31, 403. Lascelles, P. T., Taylor, W. H. ibid. 1968, 35, 63.
1365
tal circumstances in which the
two sets
Departments of Chemical Pathology, National Hospital, Queen Square, London W.C.1, and United Liverpool Hospitals, Liverpool, L3 5RT.
P. T. LASCELLES W. H. TAYLOR.
POLYPLOIDY SIR,-Polyploidy of human lymphocytes has been observed in malignant disease,1,2 after irradiation, and following administration of various drugs.3,4 Abnormalities of E-group chromosomes and increases in chromosomal breaks, on the other hand, are associated with disturbances in production of immunoglobulins.5-8 We have observed chromosomal polyploidy in a patient with immunoglobulin
deficiency. The patient was a 29-year-old farm labourer who had a history of frequent pulmonary infection since the age of 5, when he had whooping-cough. In March, 1968, he was admitted to hospital with pneumonia. He was of short stature (height 150 cm.), his i.Q. was 63, and he had had an operation on his right hand for polydactyly. The peripheralleucocyte count was within normal limits (lymphocytes did not exceed 15%). In bone-marrow smears plasma-cells were totally absent. Serum-immunoglobulin assay showed that IgA and IgM were absent, and IgG were considerably diminished. Various bacterial antibody titres were diminished or undetectable. Chromosome studies were done on 48-hour cultures of peripheral leucocytes stimulated by phytohaemagglutinin, and directly from sternal bone-marrow cells obtained by needle aspiration. Among the 33 photographed mitoses there were 8 polyploid cells, with chromosome numbers ranging from 63 to 93. The diploid cells showed a normal male (46,XY) karyotype. There were chromosomal breaks in 9 of the diploid cells, and in 3 of these chromosome number 2 was dicentric. E-group chromosomes were normal. Among the 31 karyotyped bone-marrow mitoses there were 4 polyploid cells. The diploid cells in the bonemarrow cells had normal karyotypes, with the exception of 1 with a supernumerary E-group chromosome. Further observations in other patients will be needed in order to ascertain whether this conspicuous polyploidy was related to the immunoglobulin deficiency. There is also the that polyploidy was an early sign of malignant disease. This is common in patients with immunoglobulin deficiencies,9 but so far there are no signs of lymphoma in
possibility
patient.
A detailed
description of this case will be published elsewhere. Department of Pathology, Robert Károly Hospital, M. SELLYEI. Budapest XIII. Department of Medicine, National Institute of Rheumatology, GABRIELLA KISS. Budapest III. State Human " Serum Institute, R. BACKHAUSZ. Budapest X. "
1. 2. 3.
toxicity 476) are interesting and thought-provoking. You conclude by saying that the use of chloramphenicol should be restricted to patients with typhoid fever or other gram-negative infections where no suitable alternative antibiotic is available; this statement needs some qualification. Typhoid fever is primarily a disease of developing (Aug. 30,
IMMUNOGLOBULIN DEFICIENCY AND
our
DRUGS FOR TYPHOID FEVER SIR,-Your remarks about chloramphenicol
of data have been
obtained.
Lancet, 1964, ii, 511. Bauke, J., Schöffling, K. Cancer, Philad. 1968, 22, 686. Friedman, B. I., Saenger, E. L., Kreindler, M. S. Lancet, 1964, ii, 494. 4. Nasjleti, C. E., Spencer, H. H. Cancer, Philad. 1967, 20, 31. 5. Haddad, Z. H., Allen, R. F., Towner, J. W., Wilson, M. G. Lancet, 1969, i, 678. 6. Hecht, F. ibid. p. 100. 7. Jacobs, J. C., de Capoa, A., McGilvray, E., Morse, J. H., Schullinger, J. N., Blanc, W. A., Heird, W. C., Miller, O. J., Rossen, R. D., Walzer, R. A. ibid. 1968, i, 499. 8. Hecht, F., Koler, R. D., Rigas, D. A., Dahnke, G. S., Case, M. P., Tisdale, V., Miller, R. W. ibid. 1966, ii, 1193. 9. ibid. 1969, i, 163.
p.
countries. The incidence of enteric fever in West Pakistan has been estimated to be about 1 %. On this basis, half a million cases probably occur in West Pakistan every year. Only six thousand of these patients are admitted to Government hospitals.1 The majority are treated outside the hospital by general practitioners. In general practice, as in the hospitals, treatment is normally instituted immediately upon clinical diagnosis of typhoid fever. Confirmation by serological tests is not awaited in most cases.2 Serological and blood-culture facilities are not widely available in the countries where typhoid is most prevalent, and where available they are expensive and time-consuming. Under these circumstances, a large number of patients, supposedly having typhoid fever, are exposed to the hazards of
chloramphenicol. Furazolidone, a nitrofuran derivative, has recently been extensively tried in the treatment of typhoid fever in India,3-1o Pakistan,11,12 and elsewhere.13,14 The results have been
encouraging. Furazolidone has also been used for disorders of the gastrointestinal tract for more than 8 years, and no serious toxic reactions have been reported. Under these circumstances, I feel that furazolidone should be tried first in all cases of typhoid fever. I think you will agree that its use in the clinically diagnosed cases of typhoid fever will serve to protect patients from unnecessary exposure to In my opinion, chloramphenicol should patients with typhoid fever who require parenteral therapy or who fail to respond to furazolidone. No cross-resistance has been reported between the two drugs.
chloramphenicol.
be reserved for
Jinnah Postgraduate Medical Centre, Karachi, Pakistan.
ANWAR MASOOD.
ŒSOPHAGEAL CANCER IN AFRICA
SIR,-It is possible that the apparent great increase in cancer of the oesophagus in parts of Africa, to which you refer in your annotation (Nov. 29, p. 1178), is due partly to increased longevity. Palmar and plantar keratosis, which I suspect is common in Africa, is one condition associated with cancer of the oesophagus in late middle life. (It is a long time since I worked in West and South Africa, and I did not know about this familial disease syndrome at the time, but I remember wondering why some Africans had thickened palms though they were not employed in hard manual labour.) Even in Britain there are no good records of the diseases of past generations. I believe that when such records become available, certain forms of cancer will be found to be associated with developmental anomalies. In relation to palmar keratosis in Africans, it would be important to Awan, A. H. Pakist. J. med. Res. 1969, 8, 91. Hameedi, A. A. J. Pakist. med. Ass. 1964, 14, 745. Chakraborty, G. Indian J. Pœdiat. 1961, 28, 164. Chakraborty, G. J. Indian med. Ass. 1958, 41, 10. Damany, S. J., Bilgi, C. L. J. Ass. Phys. India, 1968, 16, 669. Jaffari, S. M. H. Punjab med. J. 1966, 15, 423. Kamat, S. A. J. Am. med. Ass. 1968, 206, 2745. Luhar, P. M. Clin. Trials J. 1967, 4, 816. Mehta, M. R. Indian Practitioner, 1967, 20, 327. Solanki, S. V., Kabrawala, V. N. Saurashtra Univ. J. 1968, I, 155. Khan, N. M. Medicus, 1969, 38, 258. Razzaq, A., Hayee, A. Pakist. J. med. Res. (in the press). Musgrave, M. E., Brownlow, W. J. Cited by Jaffari, S. M. H. Punjab med. J. 1966, 15, 423. 14. Paul, M. R. Antibiotics Chemother. 1960, 10, 231. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.