Annals of Tropical Medicine & Parasitology
ISSN: 0003-4983 (Print) 1364-8594 (Online) Journal homepage: http://www.tandfonline.com/loi/ypgh19
The urinary excretion of chloroquine in different ethnic groups D. A. Price Evans, K. A. Fletcher & J. D. Baty To cite this article: D. A. Price Evans, K. A. Fletcher & J. D. Baty (1979) The urinary excretion of chloroquine in different ethnic groups, Annals of Tropical Medicine & Parasitology, 73:1, 11-17, DOI: 10.1080/00034983.1979.11687220 To link to this article: http://dx.doi.org/10.1080/00034983.1979.11687220
Published online: 11 Mar 2016.
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Citing articles: 9 View citing articles
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Date: 21 September 2017, At: 19:59
Annals of Tropical Medicine and Parasitology, Vol. 73, No. 1 (1979)
The urinary excretion of chloroquine in different ethnic groups BY D. A. PRICE EVANS
Downloaded by [Australian Catholic University] at 19:59 21 September 2017
Nu.ffield Unit of Medical Genetics, Department of Medicine, University of Liverpool, Liverpool L69 3BX K. A. FLETCHER
Department of Tropical Medicine, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA AND
J. D.
BATY*
Nuffield Unit of Medical Genetics, Department of Medicine, University of Liverpool, Liverpool L69 3BX Received 1 March 1978 Although chloroquine, 7-chloro-4-(4' -diethylamino-1' -methy1buty1amino) quinoline, has been used throughout the world as an anti-malarial drug for over 30 years, there have been no studies to investigate whether its handling and excretion varies in different popu1ations. Usually, for the radical cure of falciparum malaria, a standard regimen of 1·5-2·4 g of chloroquine is administered over a three-day period on a global basis, although in partially irrunune populations a single dose of600 mg is usually sufficient (World Health Organization, 1967). Chloroquine is rapidly and almost completely absorbed from the gastrointestinal tract. Approximately 55% of the drug is bound to non-diffusable plasma constituents and it is avidly taken up and deposited in many tissues of the body. Excretion of chloroquine is quite slow (Goodman and Gilman, 1975). Studies so far indicate that chloroquine is only slowly metabolized, the main metabolite being the mono de-ethylated compound, desethyl-chloroquine (McChesney, Fasco and Banks, 1967; Kuroda, 1962). Preliminary studies in man and monkeys in our laboratories using gas chromatography/mass spectrometry support these findings (Fletcher, Baty, Evans and Gilles, 1975). In view of the lack of information on comparative excretion rates of chloroquine in different ethnic groups, healthy volunteers of various racial origins were studied under controlled conditions of diet and fluid intake. Ancillary studies were carried out to ascertain ( 1) whether in vivo acidification of the urine causing an increased excretion of the basic drug would enhance the interpretation of the test, and (2) if additional estimation of the de-ethylated metabolite of chloroquine would give any further useful information. After some preliminary experiments, a single standardized excretion test was performed on several ethnic groups and the results form the basis of this communication. *Present address: Department of Chemical Medicine, Ninewells Hospital, University of Dundee, Dundee, Scotland. 0003-4983/79/010011 +07 SOI.00/0
© 1979 Liverpool School of Tropical Medicine
12
EXCRETION OF CHLOROQUINE IN DIFFERENT ETHNIC GROUPS
Downloaded by [Australian Catholic University] at 19:59 21 September 2017
MATERIALS AND METHODS Urinary creatinine was estimated by the alkaline picrate method described by Owen, Iggo, Scandrett and Stewart (1954) and Ralston (1955). Chloroquine in urine was estimated by a modification of the method described by Brodie, Udenfriend, Dill and Chenkin (1947). The essential details are as follows: after collection urine specimens were acidified to pH 2·0 with HCl (1/l,vjv, cone. with water), with no significant increase in volume ( < 1%) . A 5 ml aliquot of the acidified urine specimen was extracted with 10 ml re-distilled n-heptane (BDH Chemicals Ltd., England) by vortex mixing for two minutes. On the basis of the creatinine concentration, the urine was suitably diluted if necessary to bring the fluorimetric reading within the range of the standards. The organic phase was then discarded. To the aqueous phase was added 1 ml 4 M ammonium hydroxide and 0·5 ml 0·1 M ammonium hydroxide in 0·2 M ammonium chloride, which took the pH to 9·3. The aqueous phase was again extracted with 10 ml n-heptane by vortex mixing for two minutes. A 5 ml aliquot of the heptane layer was then extracted with 2 ml 0·01 N HCI. A 1 ml aliquot of the acid phase was then added to 1 ml 0·33 N NaOH and I ml 0·6 M boric acid in 0·6 M potassium chloride, the fluorescence of which was then read in a Perkin-Elmer MPF-3 recording spectrofluorimeter with excitation of340 nm and emission of400 nm. A range of chloroquine standards from 2·5 ~g to 15·0 ~g/ ml in 0·01 N HCI were prepared and 5 ml aliquots treated identically to the urine samples. Duplicate estimations of chloroquine in the urine samples and the standard solutions were performed in this study. Results were discarded and estimations repeated if duplicates varied by more than 5%. All glassware used throughout the technique was washed in chromic acid ('Chromerge', Camlab. Ltd., England), rinsed with tap water and finally with glass-distilled water, using a standard procedure. The relative response of desethyl-chloroquine and chloroquine on the gas liquid chromatography (GLC) was determined by extracting known weights of the two compounds from drug-free urine (Pye model 104 gas chromatograph, using a glass column packed with 3% OV 17 on Gas Chrom.-Q, column temperature 230°). The relative GLC peak height response of desethyl-chloroquine was found to be 75% of that due to chloroquine over a wide concentration range. The actual concentration of the metabolite in a patient's sample was then determined by interpolation between the GLC peak heights, using the chloroquine concentration determined by spectrofluorimetry. The metabolite was shown not to interfere with the spectrofluorimetric assay. In one experiment, ammonium chloride was administered to two volunteers to produce acidification of the urine. On the day prior to the test 5·5 g NH 4Cl was given over a ninehour period. A further 1·5 g was given the next day, one hour before the chloroquine tablets were swallowed. All other subjects were studied using the standard test described below. The standard chloroquine excretion test was performed on healthy volunteers who were not on medication and whose alcohol consumption was not excessive. The test was started after a light fat-free breakfast. The bladder was emptied and the urine saved. Four 150 mg tablets of chloroquine base ('Nivaquine 200', May and Baker Ltd., England) were crushed in the mouth and swallowed with one pint of water. At hourly intervals for the next seven hours the bladder was completely emptied and a pint of water was supplied. The timing of specimen-taking was rigorously controlled. Four hours after the chloroquine tablets, a light fat-free lunch was given with one pint of unsweetened tea as an alternative to the usual water. Volunteers were not forced to drink one whole pint each hour, but consumed as great a volume as possible without causing discomfort in order to maintain a good urine flow. The pH of all urine samples was measured immediately after voiding. [The pH of urine samples from North Europeans, South-east Asians and Sudanese (who
PRICE EVANS ET AL.
13
Downloaded by [Australian Catholic University] at 19:59 21 September 2017
were all tested in Liverpool) was determined with a direct reading pH meter (Electronic Instruments Ltd., England). The Gambian urine specimens were, however, collected in The Gambia and could only be tested with pH papers.] The urines were then acidified to pH 2·0, their volumes measured and they were then stored at -20°C until chemical analyses were carried out.
Experimental Subjects Subjects other than the Gambians ( 11 North Europeans; seven South-east Asians; four Sudanese) were tested in Liverpool and consisted of healthy doctors, dentists and medical students. The 11 Gambian subjects were tested in The Gambia and consisted of healthy laboratory staff and nurses. Statistical Methods These were standard. RESULTS
Assessment of Effect of pH on Chloroquine Excretion Chloroquine is a base and so is excreted to a greater extent the lower the pH of the urine. To assess this aspect, two British white student volunteers were given a one-day chloroquine test involving a single dose of 600 mg base and hourly urine collections for seven hours thereafter without ammonium chloride, and another two British white student volunteers were given the identical test with ammonium chloride to acidifY the urine (see Appendix 1 for schedule). The results are shown in Table 1 and indicate clearly that a low urinary pH TABLE 1
Effect of urinary pH on chloroquine and desethyl-chloroquine excretion in British white subjects Volunteer number 1 (with arnmon. chloride) 2 (with ammon. chloride) 3 No arnmon. chloride 4 No arnmon. chloride
Wt. chloroquine excreted in seven hours following drug ingestion (mg)
deset~l-chloroquine
pH range
34·6 25·7 16·6 13-6
4·0 2-7 3·3 3·5
4·7-5·3 5·5-6·7 5·1-6·8
Mean%
4·~5·4
results in the excretion of a larger proportion of a single dose of the compound. However it was also apparent that the schedule in Appendix 1 would be unacceptable for any largescale population screening experiment because of the nausea, abdominal discomfort and looseness of the bowels which it produced.
luitial Experiment to Compare Two Ethnic Groups Two Thai subjects were treated with the same protocol as the two British subjects mentioned above who did not receive ammonium chloride (see Appendix 2); the dosage in terms of mg chloroquine per kg body weight varied from 9-43 to 11·32 mg per kg. The pH of the freshly passed urine was in the same range for all four subjects, namely 5·1 to 6·8. The excretion of chloroquine in terms of mg per kg body weight was 0·26 and 0·21 for the two British and 0·58 and 0·75 for the two Thai subjects. These results suggested that it would be worth doing a survey of inter-ethnic variability of chloroquine excretion following a single standard oral dose even though it was not feasible to exert rigid control over the urinary pH.
Downloaded by [Australian Catholic University] at 19:59 21 September 2017
14
EXCRETION OF CHLOROQUINE IN DIFFERENT ETHNIC GROUPS
lnter-etlmic Comparisons The weight of chloroquine excreted in the seven hours following the ingestion of a standard oral dose of 600 mg is shown for the four ethnic groups studied in the Fig. A one-way analysis of variance shows F = 25·7 with degrees of freedom (d.f.) 3 and 29, PO·lO, indicating no significant diversity between the four populations. The correlation coefficient for weights of chloroquine and creatinine excreted was 0·07 with d.f. 31, t = 0·40, P>O·lO, indicating absence of significant correlation. Examination of the Fig. reveals that the chloroquine excretion of the S.E. Asians (including Chinese) is considerably higher than that of the other three ethnic groups. Inter-ethnic Distribution of Body Weights and Body Heights An analysis of variance of body weights yielded F = 4·56 with d.f. 3 and 29, PO·lO). The inter-ethnic distribution of height and its relationship to chloroquine excretion was very similar to weight. Urine Volume For each hourly interval following chloroquine ingestion a correlation coefficient wa~ calculated between urine volume (x) and weight chloroquine excreted (y), pooling all four ethnic groups. Only one significant correlation coefficient was obtained, namely that of the first time interval (r = 0·45, t = 2·8, d.f. 31, P
ISSN: 0003-4983 (Print) 1364-8594 (Online) Journal homepage: http://www.tandfonline.com/loi/ypgh19
The urinary excretion of chloroquine in different ethnic groups D. A. Price Evans, K. A. Fletcher & J. D. Baty To cite this article: D. A. Price Evans, K. A. Fletcher & J. D. Baty (1979) The urinary excretion of chloroquine in different ethnic groups, Annals of Tropical Medicine & Parasitology, 73:1, 11-17, DOI: 10.1080/00034983.1979.11687220 To link to this article: http://dx.doi.org/10.1080/00034983.1979.11687220
Published online: 11 Mar 2016.
Submit your article to this journal
View related articles
Citing articles: 9 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ypgh19 Download by: [Australian Catholic University]
Date: 21 September 2017, At: 19:59
Annals of Tropical Medicine and Parasitology, Vol. 73, No. 1 (1979)
The urinary excretion of chloroquine in different ethnic groups BY D. A. PRICE EVANS
Downloaded by [Australian Catholic University] at 19:59 21 September 2017
Nu.ffield Unit of Medical Genetics, Department of Medicine, University of Liverpool, Liverpool L69 3BX K. A. FLETCHER
Department of Tropical Medicine, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA AND
J. D.
BATY*
Nuffield Unit of Medical Genetics, Department of Medicine, University of Liverpool, Liverpool L69 3BX Received 1 March 1978 Although chloroquine, 7-chloro-4-(4' -diethylamino-1' -methy1buty1amino) quinoline, has been used throughout the world as an anti-malarial drug for over 30 years, there have been no studies to investigate whether its handling and excretion varies in different popu1ations. Usually, for the radical cure of falciparum malaria, a standard regimen of 1·5-2·4 g of chloroquine is administered over a three-day period on a global basis, although in partially irrunune populations a single dose of600 mg is usually sufficient (World Health Organization, 1967). Chloroquine is rapidly and almost completely absorbed from the gastrointestinal tract. Approximately 55% of the drug is bound to non-diffusable plasma constituents and it is avidly taken up and deposited in many tissues of the body. Excretion of chloroquine is quite slow (Goodman and Gilman, 1975). Studies so far indicate that chloroquine is only slowly metabolized, the main metabolite being the mono de-ethylated compound, desethyl-chloroquine (McChesney, Fasco and Banks, 1967; Kuroda, 1962). Preliminary studies in man and monkeys in our laboratories using gas chromatography/mass spectrometry support these findings (Fletcher, Baty, Evans and Gilles, 1975). In view of the lack of information on comparative excretion rates of chloroquine in different ethnic groups, healthy volunteers of various racial origins were studied under controlled conditions of diet and fluid intake. Ancillary studies were carried out to ascertain ( 1) whether in vivo acidification of the urine causing an increased excretion of the basic drug would enhance the interpretation of the test, and (2) if additional estimation of the de-ethylated metabolite of chloroquine would give any further useful information. After some preliminary experiments, a single standardized excretion test was performed on several ethnic groups and the results form the basis of this communication. *Present address: Department of Chemical Medicine, Ninewells Hospital, University of Dundee, Dundee, Scotland. 0003-4983/79/010011 +07 SOI.00/0
© 1979 Liverpool School of Tropical Medicine
12
EXCRETION OF CHLOROQUINE IN DIFFERENT ETHNIC GROUPS
Downloaded by [Australian Catholic University] at 19:59 21 September 2017
MATERIALS AND METHODS Urinary creatinine was estimated by the alkaline picrate method described by Owen, Iggo, Scandrett and Stewart (1954) and Ralston (1955). Chloroquine in urine was estimated by a modification of the method described by Brodie, Udenfriend, Dill and Chenkin (1947). The essential details are as follows: after collection urine specimens were acidified to pH 2·0 with HCl (1/l,vjv, cone. with water), with no significant increase in volume ( < 1%) . A 5 ml aliquot of the acidified urine specimen was extracted with 10 ml re-distilled n-heptane (BDH Chemicals Ltd., England) by vortex mixing for two minutes. On the basis of the creatinine concentration, the urine was suitably diluted if necessary to bring the fluorimetric reading within the range of the standards. The organic phase was then discarded. To the aqueous phase was added 1 ml 4 M ammonium hydroxide and 0·5 ml 0·1 M ammonium hydroxide in 0·2 M ammonium chloride, which took the pH to 9·3. The aqueous phase was again extracted with 10 ml n-heptane by vortex mixing for two minutes. A 5 ml aliquot of the heptane layer was then extracted with 2 ml 0·01 N HCI. A 1 ml aliquot of the acid phase was then added to 1 ml 0·33 N NaOH and I ml 0·6 M boric acid in 0·6 M potassium chloride, the fluorescence of which was then read in a Perkin-Elmer MPF-3 recording spectrofluorimeter with excitation of340 nm and emission of400 nm. A range of chloroquine standards from 2·5 ~g to 15·0 ~g/ ml in 0·01 N HCI were prepared and 5 ml aliquots treated identically to the urine samples. Duplicate estimations of chloroquine in the urine samples and the standard solutions were performed in this study. Results were discarded and estimations repeated if duplicates varied by more than 5%. All glassware used throughout the technique was washed in chromic acid ('Chromerge', Camlab. Ltd., England), rinsed with tap water and finally with glass-distilled water, using a standard procedure. The relative response of desethyl-chloroquine and chloroquine on the gas liquid chromatography (GLC) was determined by extracting known weights of the two compounds from drug-free urine (Pye model 104 gas chromatograph, using a glass column packed with 3% OV 17 on Gas Chrom.-Q, column temperature 230°). The relative GLC peak height response of desethyl-chloroquine was found to be 75% of that due to chloroquine over a wide concentration range. The actual concentration of the metabolite in a patient's sample was then determined by interpolation between the GLC peak heights, using the chloroquine concentration determined by spectrofluorimetry. The metabolite was shown not to interfere with the spectrofluorimetric assay. In one experiment, ammonium chloride was administered to two volunteers to produce acidification of the urine. On the day prior to the test 5·5 g NH 4Cl was given over a ninehour period. A further 1·5 g was given the next day, one hour before the chloroquine tablets were swallowed. All other subjects were studied using the standard test described below. The standard chloroquine excretion test was performed on healthy volunteers who were not on medication and whose alcohol consumption was not excessive. The test was started after a light fat-free breakfast. The bladder was emptied and the urine saved. Four 150 mg tablets of chloroquine base ('Nivaquine 200', May and Baker Ltd., England) were crushed in the mouth and swallowed with one pint of water. At hourly intervals for the next seven hours the bladder was completely emptied and a pint of water was supplied. The timing of specimen-taking was rigorously controlled. Four hours after the chloroquine tablets, a light fat-free lunch was given with one pint of unsweetened tea as an alternative to the usual water. Volunteers were not forced to drink one whole pint each hour, but consumed as great a volume as possible without causing discomfort in order to maintain a good urine flow. The pH of all urine samples was measured immediately after voiding. [The pH of urine samples from North Europeans, South-east Asians and Sudanese (who
PRICE EVANS ET AL.
13
Downloaded by [Australian Catholic University] at 19:59 21 September 2017
were all tested in Liverpool) was determined with a direct reading pH meter (Electronic Instruments Ltd., England). The Gambian urine specimens were, however, collected in The Gambia and could only be tested with pH papers.] The urines were then acidified to pH 2·0, their volumes measured and they were then stored at -20°C until chemical analyses were carried out.
Experimental Subjects Subjects other than the Gambians ( 11 North Europeans; seven South-east Asians; four Sudanese) were tested in Liverpool and consisted of healthy doctors, dentists and medical students. The 11 Gambian subjects were tested in The Gambia and consisted of healthy laboratory staff and nurses. Statistical Methods These were standard. RESULTS
Assessment of Effect of pH on Chloroquine Excretion Chloroquine is a base and so is excreted to a greater extent the lower the pH of the urine. To assess this aspect, two British white student volunteers were given a one-day chloroquine test involving a single dose of 600 mg base and hourly urine collections for seven hours thereafter without ammonium chloride, and another two British white student volunteers were given the identical test with ammonium chloride to acidifY the urine (see Appendix 1 for schedule). The results are shown in Table 1 and indicate clearly that a low urinary pH TABLE 1
Effect of urinary pH on chloroquine and desethyl-chloroquine excretion in British white subjects Volunteer number 1 (with arnmon. chloride) 2 (with ammon. chloride) 3 No arnmon. chloride 4 No arnmon. chloride
Wt. chloroquine excreted in seven hours following drug ingestion (mg)
deset~l-chloroquine
pH range
34·6 25·7 16·6 13-6
4·0 2-7 3·3 3·5
4·7-5·3 5·5-6·7 5·1-6·8
Mean%
4·~5·4
results in the excretion of a larger proportion of a single dose of the compound. However it was also apparent that the schedule in Appendix 1 would be unacceptable for any largescale population screening experiment because of the nausea, abdominal discomfort and looseness of the bowels which it produced.
luitial Experiment to Compare Two Ethnic Groups Two Thai subjects were treated with the same protocol as the two British subjects mentioned above who did not receive ammonium chloride (see Appendix 2); the dosage in terms of mg chloroquine per kg body weight varied from 9-43 to 11·32 mg per kg. The pH of the freshly passed urine was in the same range for all four subjects, namely 5·1 to 6·8. The excretion of chloroquine in terms of mg per kg body weight was 0·26 and 0·21 for the two British and 0·58 and 0·75 for the two Thai subjects. These results suggested that it would be worth doing a survey of inter-ethnic variability of chloroquine excretion following a single standard oral dose even though it was not feasible to exert rigid control over the urinary pH.
Downloaded by [Australian Catholic University] at 19:59 21 September 2017
14
EXCRETION OF CHLOROQUINE IN DIFFERENT ETHNIC GROUPS
lnter-etlmic Comparisons The weight of chloroquine excreted in the seven hours following the ingestion of a standard oral dose of 600 mg is shown for the four ethnic groups studied in the Fig. A one-way analysis of variance shows F = 25·7 with degrees of freedom (d.f.) 3 and 29, PO·lO, indicating no significant diversity between the four populations. The correlation coefficient for weights of chloroquine and creatinine excreted was 0·07 with d.f. 31, t = 0·40, P>O·lO, indicating absence of significant correlation. Examination of the Fig. reveals that the chloroquine excretion of the S.E. Asians (including Chinese) is considerably higher than that of the other three ethnic groups. Inter-ethnic Distribution of Body Weights and Body Heights An analysis of variance of body weights yielded F = 4·56 with d.f. 3 and 29, PO·lO). The inter-ethnic distribution of height and its relationship to chloroquine excretion was very similar to weight. Urine Volume For each hourly interval following chloroquine ingestion a correlation coefficient wa~ calculated between urine volume (x) and weight chloroquine excreted (y), pooling all four ethnic groups. Only one significant correlation coefficient was obtained, namely that of the first time interval (r = 0·45, t = 2·8, d.f. 31, P
