Chloramphenicol
metabolism
protein-calorie S. Mehia,
H.
K.
malnutrition1’
Kalsi,
S. Jayaraman
ABSTRACT
The
malnourished mg/kg
plasma
children
body
weight.
in malnourished
and Plasma
children
from
the
mal.
These
observations
tern was
of the in the
drug form
plasma,
nourished
children.
transferase
and
biosynthesis
V. S. Mathur
levels
and
four
normal
peak
levels
compared
30 hours
or more point
Journal
excretion after
were
to the
achieved
2-4
normal. rate
of chloramphenicol oral
In two levels
children were
the
liver
detected.
also
children
later took
Am.
Nutrition
28: SEPTEMBER
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/9/977/4716492 by University of California, Berkeley/LBL user on 28 July 2018
tissue
observation
J. Clin.
Nuir.
studied
of a single
and
were
much
with
in the
to clear 12 hours
subjected
also
points
28: 977-981,
of 25
liver.
The
to assay
higher the
drug
in the
nor-
excretion
75-85% of the drug 35-55% was conjugated
was
in ten
dose
1.5 or 2 times
longer
compared
of biotransformation
biopsy
This
was
administration
hours
They
in malnournished
to a slower
of chloramphenicol.
of Clinical
urinary children
and its metabolite lends support to this hypothesis. of conjugated fraction in the normal while only low
with
2
and
A remarkable accumulation of knowledge of the ways in which an organism handles drugs has led to the elucidation of such processes as biotransformation, distribution and disposal of drugs. An understanding of drug disposition has allowed a more rational basis for employing drug therapy. It is now widely recognized that animals of varying maturity exhibit differences in drug handling. Of equal importance may be the influence of pathologic states on the drug metabolism in the developing individual. This paper describes efforts to study the effects of one of the major health problems of children, namely, protein-calorie malnutrition (PCM), on drug metabolism. Children suffering from PCM have a high incidence of intercurrent infections and require frequent treatment with antibiotics. In India, chloramphenicol is commonly used for gastrointestinal and respiratory infections. A rational use of the drug necessitates a study of its disposition in PCM. Chloramphenicol is highly lipid soluble and is absorbed and distributed in the body with great ease. It is normally biotransformed in the liver by conjugation to glucuronide. Practically 90% of the drug excreted in the urine by an adult is in the form of a monoglucuronide. Hence it provides a good model to study phase II reaction (conjugation). Irreversible toxicity to bone marrow with The American
in children
patexcreted in mal-
of bilirubin-UDP
to an alteration
in the
rate
of
1975.
chloramphenicol occurs in only I in 50,000 to 100,000 individuals. In contrast nearly 10% of children hospitalized receive this drug for typhoid fever, shigellosis and a variety of other infections. Faced as we are, with extremely poor knowledge about the various biological aberrations that might exist in protein -calorie malnutrition in relation to drug handling, it is imperative to learn such changes as may have direct influence on dosage and spacing of administration of drugs. It is particularly important to avoid over- or underdosage and consequent toxicity or resistance to treatment. The problem gets doubly complicated by the fact that another species of animal may not provide the information which can be easily extrapolated. Permission to carry out these investigations was granted by Indian Council of Medical Research. The present study presents the preliminary data on absorption, metabolism and excretion of single doses of chloramphenicol in children suffering from PCM. Method Ten infants
of study infants between
suffering from the ages of
PCM and 6 months
four and
3
normal years
‘From the Department of Pediatrics and Pharmacology, Postgraduate Institute of Medical Education and Research, Chandigarh, India. ‘Work supported by a grant from Indian Council of Medical
Research.
1975.
pp.
977
981.
Printed
in
U.S.A.
977
MEHTA
978 constitute
the
subjects
of the
study.
They
any dehydration or infection at the time had not been on antibiotic therapy for prior to the study. Their clinical profile
did
not
ET AL.
exhibit
of the study and at least a v.eek is presented in
Table I. The degree
of malnutrition was graded using body in comparison to the standard expected weight (50th percentile of the Harvard Scale was used as the standard weight). Three infants had body weights more than 60% of the standard weight for age. None of these showed edema of feet or face. Seven children had body weights less than 60% of the expected. None of them had weight
0 40
Results In the four normal children, peak plasma levels were achieved 2 hours after administration of oral drug. Peak levels ranged from 15.0 to 20.4 zg/ml with a mean of 17.8 ig/ml. At 12 hours, chloramphenicol values fell to a low level varying from 1.8 to 3.1 jzg/ml and none was detected thereafter. A TABLE Age and
I grade
of
Age, months
in 10 subjects
is based
Ill
3
4 2 4 of PCM
Grades
1 2 4
-
on
body
+ IV
weight
for
age
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/9/977/4716492 by University of California, Berkeley/LBL user on 28 July 2018
MALE
10
5! 4
TIME
FIG.
I.
IN
HOURS
Plasma chloramphenicol of the drug-25 mg
administration
levels drug/kg
after oral body weight.
600
500
UNCHANGED
CHLORAMPHENICOL CONJUGATED
FORM
400
z 0
300
z 200 E
a,
2
4
S
2
24
HOURS
FIG. normal
2.
Excretion
child
of
chloramphenicol
in
urine
in
of 3 years.
representative graph is presented in Fig. 1 as obtained in a 3-year-old male. After administration of a single dose of 300 mg of chloramphenicol palmitate, the highest peak level of 17.6 zg/ml was attained in 2 hours and at 12 hours the level had fallen to 3.1 zg/ml. No drug could be detected in subsequent samples. In the urine, highest concentration was obtained in the first 4 hours (approx 500 zg/ml) and they gradually declined. Only about 40 tg/ml was detected in the last sample collected at 30 hours. The conjugated form of drug accounted for 75-80% of the drug excreted (Fig. 2). Plasma
Grades I + II
Total no.
6-12 13-24 25-36 Grading
PCM
3 YEARS
20
gross edema. Three had edema of feet only. Permission to carry out the study was obtained from the parents. Five hours prior to the study, food was witheld from each subject. Bladder was evacuated and the urine sample saved as control. Chloramphenicol palmitate (Parke, Davis) in a fine suspension was administered, orally in a single dose of 25 mg/kg body weight after withdrawl of a control sample of blood. Subsequently heparinized blood samples were collected at I -, 2-, 4-, 8-, 12-, 24- and 30-hour intervals. A collection bag for urine was affixed and the urine excreted during the above intervals was collected in separate containers. Drug estimations were carried out on plasma and urine by the method of Levine and Fischback. In the urine, unchanged drug was estimated in ethyl acetate extract. Chloramphenicol and the metabolites were determined together using urine without extraction. Each sample was examined in duplicate. The sensitivity of the method is such that it can determine as low as 0.5 pg/mI of drug in biological fluids. Recovery of drug from the fluids was complete within ± 1%. Liver biopsy was carried out in two children suffering from Grade II malnutrition with the help of Menghini needle size (0.4 x lin.). The liver tissue was subjected to assay of activity of bilirubin-UDP transferase enzyme by the method of Black and Billing (13). The results were expressed as milligram of bilirubin conjugated per gram of liver tissue.
NORMAL
30
chloramphenicol
levels
in PCM
After the oral administration of chloramphenicol, the peak levels were reached in 4-6 hours in seven children. In three, the peak levels were reached by 2 hours. The peak levels ranged from 17 zg/ml to 52 zg/ml with a mean of 21.3. All levels were in the effec-
CHLORAMPHENICOL
METABOLISM
tive therapeutic levels, namely above 10 ig/ ml. Hence the dose of 25 mg/kg was a suitable one to use for therapeutic effectiveness. Five children were studied a second time after undergoing nutritional rehabilitation for 4-8 weeks. During this period, their body weights had improved by 8-10%. Figures 3 and 4 show the responses of plasma values initially and after treatment, the normal curve is shown for comparison. Three obvious findings are: 1) Peak levels were achieved later than normal in children with PCM. The interval decreased with nutritional improvement. 2) Peak levels were initially high but became lower after rehabilitation. 3) Drug was cleared slowly at first; this improved after
excretion
979
40
-
NORMAL
30
3 YEARS
MALE
TIME
IN
HOURS
4
30
-
AP
3 YEARS
F WITH
-
PCM
GRADE
III
P(M
/TT:I:G1DPM
S TIME
FIG.
4.
2 IN
24
levels after oral body weight.
500
E__]
400
I
f]
in PCM
3’.
HOURS
Plasma chloramphenicol of drug-25 mg drug/kg
administration
rehabilitation. It was also apparent that the abnormal pattern started to improve while the recovery in body weight was not yet complete. Urinary
IN CHILDREN
UNCHANGED CONJUGATED
a,
Detailed information was available for five children with PCM before and after 4-8 weeks of rehabilitation. Patterns of these patients were similar. One of these is presented in the Fig. 5. Initially the drug could be recovered in samples collected at 30 hours in as high a concentration as 100 zg/ml. Only 35-55% of the drug was present in the conjugated form and the remainder was excreted unchanged in the urine. Rehabilitation of the child was associated with a significant change in the excretory pattern. Much higher concentrations were achieved during the initial period,
40
i30 \
z
20 $0
HALF LIFE 53 HR
U 24
30
4O 3O GRADE
2O U 4
.-‘GRADE
10
III PCM IIPCM
3 HR
4
4I?
4
12 TIME
IN
REHASILITATED 24
30
HOURS
FIG. 3. Plasma chloramphenicol levels after oral administration of drug-25 mg drug/kg body weight.
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/9/977/4716492 by University of California, Berkeley/LBL user on 28 July 2018
j3OO
IS
> I’) >.
200
4 2
oo
#{224}.NIL 2
4
24
4
30
FIG. amphenicol
2
24
30
HOURS
HOURS
PCM
6
GRADE
5. Excretion in urine
m
GRADE
ratio of conjugated in a malnourished
U
and child.
free
chlor-
i.e., 0-8 hours. Very small quantities could be recovered beyond 12 hours. This pattern is akin to the normal. Following rehabilitation, conjugated drug constituted 55-65% of the excreted total. The component was still less than that excreted by the normal child. The change of excretory pattern became evident even while the somatic recovery was only 8-10% in body weight. Liver biopsy was carried out in two children with grade II malnutrition and 7-8 mg of tissue were obtained. The activity of bilirubin-UDP transferase was 0.14 and 0.24 mg bilirubin conjugated/g of liver tissue. The values in normal adults from the same laboratory range between 0.45 to 0.95 mg/g of tissue. The values for normal children have not been established.
980
MEHTA
Discussion Observation in the normal infants showed that highest plasma levels of chloramphenicol was reached in 2 hours and it fell to a trace level by 12 hours. The pattern was very similar to that observed by Kelly and coworkers (3) in children who were given 22 mg/kg single dose and it also resembled the one observed in adults administered a 2-g single dose (10). Hence chloramphenicol palmitate in a fine suspension was easily absorbed and the pattern of decay was similar to that seen in adults receiving crystalline chloramphenicol. The children suffering from PCM showed three significant features in the plasma curve in response to oral administration of the drug. 1) Peak plasma levels were generally attained in 4 hours or more in contrast to 2 hours in the normal. 2) The peak plasma levels were generally higher in children suffering from PCM than in the controls. 3) The drug took much longer to clear from the plasma of malnourished children. Furthermore all three features tended to revert to a normal pattern in the five children who were restudied after nutritional rehabilitation. The longer time taken to achieve the peak level may be due to slower absorption. Sixty to eighty percent of children suffering from PCM of the severity studied by us show evidence of inability to absorb nutrients such as fats, carbohydrates and vitamin B52 (2). Abnormalities of the mucosa of the small bowel in the form of villous atrophy have been well described (2, 8). Under such conditions it is conceivable that chloramphenicol is not absorbed rapidly. The peak level of drug in plasma, however, is higher in malnourished infants. This cannot be explained on the basis of defective absorption. As drug absorption begins, so do the processes of distribution, binding to plasma proteins, biotransformation and excretion. The plasma level reflects the total influence of all processes in combination. Disturbances in any of these processes could result in higher plasma levels in PCM. In infants with PCM, it took longer for the drug to clear from blood. An interference
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/9/977/4716492 by University of California, Berkeley/LBL user on 28 July 2018
ET AL. with distribution or protein binding cannot be explained easily by both of these phenomena occurring simultaneously. In normal adults, the drug is bound to plasma albumin to the extent of6O%. In PCM plasma albumin levels fall. The bound form should therefore fall also. Under such circumstances the plasma clearance should not be delayed. However, we have no information at present on the exact nature and extent ofprotein binding in PCM. Delayed clearance of the drug as well as the higher peak levels in plasma can result from either a slower biotransformation or a slower renal clearance. Studies on renal function in PCM (personal communication, 0. P. Ghai) did not reveal any significant change either in glomerular filtration rate or in tubular reabsorption. The unchanged chloramphenicol is chiefly dependent on glomerular filtration and conjugated form on tubular secretion for elimination (4). Renal excretory mechanism may not be responsible for the behaviour of plasma levels. This area needs more work for complete elucidation. In PCM changes in liver cells have long been documented. In addition to gross histologic changes, electron-microscopic changes have been described in the endoplasmic reticulum in humans (12) as well as in experimental animals (6, 7). Most microsomal enzymes involved in metabolism of drugs reside in the endoplasmic reticulum. It is therefore possible that alteration in biotransformation of chloramphenicol by the hepatocyte may be responsible for the behaviour of the plasma curve. In two children, liver biopsies could be obtained before the drug was administered. Glucuronyl
transferase
was
estimated
using
bilirubin as substrate (13). The liver tissue demonstrated a reduction of the enzyme to one-half and one-third, respectively, of the normal value. Although such information gathered is preliminary, bilirubin glucuronyl transferase is probably similar to the enzyme needed for chloramphenicol conjugation. It can be postulated that a similar diminution of specific enzyme might interfere with an efficient conjugation of the drug. The data obtained from urine analysis support this postulate. Three patients with PCM excreted a large amount of unchanged drug (50-60%) in the
CHLORANIPHENICOL
METABOLISM
urine. When these children were restudied after initial rehabilitation a greater amount of conjugated drug was excreted. The pattern in the normal child showed that 75-85% of the drug in the urine was in the conjugated form. In adults nearly 90% of the drug is excreted in the conjugated form (10). The observations of the present study point to the need for a reevaluation of therapy in malnourished children. When using chloramphenicol a possibly slower rate of metabolism results in higher blood levels which take a longer time to clear. The current practice of drug administration in dosages calculated only on the basis of body weight are likely to be hazardous in a malnourished child. A reduction in dosage and wider spacing of administration may be required. Exact recommendations must be based on more extended observations and a study of multiple dose administration. Summary Metabolism of orally administered chloramphenicol palmitate was studied in ten malnourished children and four normal children between the ages of 6 months and 3 years. Drug was given in a single dose of 25 mg/kg body weight and blood samples and timed urine samples collected at 1, 2, 4, 8, 12, 24 and 30 hours. Drug was analyzed in the serum and urine. In urine the total as well as unconjugated drug was estimated. The malnourished children achieved a peak level which was delayed but higher than the drug from the normal. In the urine they excreted larger amounts of unchanged drug than the normal child. After nutritional rehabilitation these findings resembled those in normal children. The observations are explained on the basis of a slower rate of conjugation. Bilirubin-UDP transferase activity was estimated in two malnourished children and
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/9/977/4716492 by University of California, Berkeley/LBL user on 28 July 2018
981
IN CHILDREN
found to be lower than evidence lends support slow biotransformation.
in normal adults. to the hypothesis
This of
El
References 1. LEVINE, J., AND H. FISCUBACK. The chemical determination of chloramphenicol in biological materials. Antibiotics I: 59, 1951. 2. GUPTE S. P., S. NIEHTA AND B. N. S. WALIA. Small bowel function in protein-calorie malnutrition. Indian Pediat. 7:481-488, 1970. 3. KELLY R. S., A. D. HUNT AND S. G. TASHMAN. Studies on the absorption and distribution of chloramphenicol. Pediatrics 8: 362-367, 1951. 4. WEINSTEIN, L. Pharmacological Basis of Therapeutics (4th ed), edited by Goodman L. S. and A. Gilman. New York: Macmillan, 1970. 5. DELIWALA, K. E. V., AND U. K. SHETH. Chloramphenicol levels in the blood following oral administration of various chloramphenicol esters. J. Postgraduate Med., Bombay 8: 27-32, 1962. 6. OROY J. M., T. SAMORAJSKI, R. R. ZIMMERMAN ANt) P. NI. RADY. Effects of postnatal protein deficiency on weight gain, serum, proteins enzymes cholesterol and liver ultrastructure in a subhuman primate. Am. J. Pathol. 48: 796, 1966. 7. Svooi,s D., G. GRADY AND J. I-IIGGINSON. The effects of chronic protein deficiency in rats. II. Biochemical and ultrastructure changes. Lab. Invest. 15: 731, 1966. 8. BRUNSER 0., W. RElu, F. NIONCKERERG, A. MACCIONI, I. CONTRERAS AND E. TRABUCCO. Jejunal 38: 605, 9.
GLAZKO
E.
L.
biopsies 1966.
in
A. J., A. W. An
infant KINKEL,
malnutrition.
W. C.
Pediatrics ALEGNANI
ANt)
evaluation of the absorption characteristics of different chloramphenicol preparation in normal human subjects. Clin. Pharmacol. Therap. 9: 472, 1968. 10. GLAZKO A. J. Biochemical studies on chloramphenicol. II. Tissue distribution and excretion studies. J. Pharmacol. Exptl. Therap. 96: 445-459, 1949. II. AGERIAR, A. J., A. W. KINKEL AND J. C. SAMYN. Effects of polymorphism on the absorption of chloramphenicol from chloramphenicol palmitate. J. Pathol. Sci. 56: 847 -853, 1967. 12. THERON, J. J., ANt) N. LIENBERG. Some observations on fine cytology of parenchymal liver cells in kwashiorkor. J. Pathol. Bacteriol. 86: 109, 1963. 13. BLACK, NI. AND B. BILLING. Determination of bilirubin UDP transferase activity in human liver biopsies. Clin. Chem. Acta 29: 27. 1970. HOLMES.
metabolism
protein-calorie S. Mehia,
H.
K.
malnutrition1’
Kalsi,
S. Jayaraman
ABSTRACT
The
malnourished mg/kg
plasma
children
body
weight.
in malnourished
and Plasma
children
from
the
mal.
These
observations
tern was
of the in the
drug form
plasma,
nourished
children.
transferase
and
biosynthesis
V. S. Mathur
levels
and
four
normal
peak
levels
compared
30 hours
or more point
Journal
excretion after
were
to the
achieved
2-4
normal. rate
of chloramphenicol oral
In two levels
children were
the
liver
detected.
also
children
later took
Am.
Nutrition
28: SEPTEMBER
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/9/977/4716492 by University of California, Berkeley/LBL user on 28 July 2018
tissue
observation
J. Clin.
Nuir.
studied
of a single
and
were
much
with
in the
to clear 12 hours
subjected
also
points
28: 977-981,
of 25
liver.
The
to assay
higher the
drug
in the
nor-
excretion
75-85% of the drug 35-55% was conjugated
was
in ten
dose
1.5 or 2 times
longer
compared
of biotransformation
biopsy
This
was
administration
hours
They
in malnournished
to a slower
of chloramphenicol.
of Clinical
urinary children
and its metabolite lends support to this hypothesis. of conjugated fraction in the normal while only low
with
2
and
A remarkable accumulation of knowledge of the ways in which an organism handles drugs has led to the elucidation of such processes as biotransformation, distribution and disposal of drugs. An understanding of drug disposition has allowed a more rational basis for employing drug therapy. It is now widely recognized that animals of varying maturity exhibit differences in drug handling. Of equal importance may be the influence of pathologic states on the drug metabolism in the developing individual. This paper describes efforts to study the effects of one of the major health problems of children, namely, protein-calorie malnutrition (PCM), on drug metabolism. Children suffering from PCM have a high incidence of intercurrent infections and require frequent treatment with antibiotics. In India, chloramphenicol is commonly used for gastrointestinal and respiratory infections. A rational use of the drug necessitates a study of its disposition in PCM. Chloramphenicol is highly lipid soluble and is absorbed and distributed in the body with great ease. It is normally biotransformed in the liver by conjugation to glucuronide. Practically 90% of the drug excreted in the urine by an adult is in the form of a monoglucuronide. Hence it provides a good model to study phase II reaction (conjugation). Irreversible toxicity to bone marrow with The American
in children
patexcreted in mal-
of bilirubin-UDP
to an alteration
in the
rate
of
1975.
chloramphenicol occurs in only I in 50,000 to 100,000 individuals. In contrast nearly 10% of children hospitalized receive this drug for typhoid fever, shigellosis and a variety of other infections. Faced as we are, with extremely poor knowledge about the various biological aberrations that might exist in protein -calorie malnutrition in relation to drug handling, it is imperative to learn such changes as may have direct influence on dosage and spacing of administration of drugs. It is particularly important to avoid over- or underdosage and consequent toxicity or resistance to treatment. The problem gets doubly complicated by the fact that another species of animal may not provide the information which can be easily extrapolated. Permission to carry out these investigations was granted by Indian Council of Medical Research. The present study presents the preliminary data on absorption, metabolism and excretion of single doses of chloramphenicol in children suffering from PCM. Method Ten infants
of study infants between
suffering from the ages of
PCM and 6 months
four and
3
normal years
‘From the Department of Pediatrics and Pharmacology, Postgraduate Institute of Medical Education and Research, Chandigarh, India. ‘Work supported by a grant from Indian Council of Medical
Research.
1975.
pp.
977
981.
Printed
in
U.S.A.
977
MEHTA
978 constitute
the
subjects
of the
study.
They
any dehydration or infection at the time had not been on antibiotic therapy for prior to the study. Their clinical profile
did
not
ET AL.
exhibit
of the study and at least a v.eek is presented in
Table I. The degree
of malnutrition was graded using body in comparison to the standard expected weight (50th percentile of the Harvard Scale was used as the standard weight). Three infants had body weights more than 60% of the standard weight for age. None of these showed edema of feet or face. Seven children had body weights less than 60% of the expected. None of them had weight
0 40
Results In the four normal children, peak plasma levels were achieved 2 hours after administration of oral drug. Peak levels ranged from 15.0 to 20.4 zg/ml with a mean of 17.8 ig/ml. At 12 hours, chloramphenicol values fell to a low level varying from 1.8 to 3.1 jzg/ml and none was detected thereafter. A TABLE Age and
I grade
of
Age, months
in 10 subjects
is based
Ill
3
4 2 4 of PCM
Grades
1 2 4
-
on
body
+ IV
weight
for
age
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/9/977/4716492 by University of California, Berkeley/LBL user on 28 July 2018
MALE
10
5! 4
TIME
FIG.
I.
IN
HOURS
Plasma chloramphenicol of the drug-25 mg
administration
levels drug/kg
after oral body weight.
600
500
UNCHANGED
CHLORAMPHENICOL CONJUGATED
FORM
400
z 0
300
z 200 E
a,
2
4
S
2
24
HOURS
FIG. normal
2.
Excretion
child
of
chloramphenicol
in
urine
in
of 3 years.
representative graph is presented in Fig. 1 as obtained in a 3-year-old male. After administration of a single dose of 300 mg of chloramphenicol palmitate, the highest peak level of 17.6 zg/ml was attained in 2 hours and at 12 hours the level had fallen to 3.1 zg/ml. No drug could be detected in subsequent samples. In the urine, highest concentration was obtained in the first 4 hours (approx 500 zg/ml) and they gradually declined. Only about 40 tg/ml was detected in the last sample collected at 30 hours. The conjugated form of drug accounted for 75-80% of the drug excreted (Fig. 2). Plasma
Grades I + II
Total no.
6-12 13-24 25-36 Grading
PCM
3 YEARS
20
gross edema. Three had edema of feet only. Permission to carry out the study was obtained from the parents. Five hours prior to the study, food was witheld from each subject. Bladder was evacuated and the urine sample saved as control. Chloramphenicol palmitate (Parke, Davis) in a fine suspension was administered, orally in a single dose of 25 mg/kg body weight after withdrawl of a control sample of blood. Subsequently heparinized blood samples were collected at I -, 2-, 4-, 8-, 12-, 24- and 30-hour intervals. A collection bag for urine was affixed and the urine excreted during the above intervals was collected in separate containers. Drug estimations were carried out on plasma and urine by the method of Levine and Fischback. In the urine, unchanged drug was estimated in ethyl acetate extract. Chloramphenicol and the metabolites were determined together using urine without extraction. Each sample was examined in duplicate. The sensitivity of the method is such that it can determine as low as 0.5 pg/mI of drug in biological fluids. Recovery of drug from the fluids was complete within ± 1%. Liver biopsy was carried out in two children suffering from Grade II malnutrition with the help of Menghini needle size (0.4 x lin.). The liver tissue was subjected to assay of activity of bilirubin-UDP transferase enzyme by the method of Black and Billing (13). The results were expressed as milligram of bilirubin conjugated per gram of liver tissue.
NORMAL
30
chloramphenicol
levels
in PCM
After the oral administration of chloramphenicol, the peak levels were reached in 4-6 hours in seven children. In three, the peak levels were reached by 2 hours. The peak levels ranged from 17 zg/ml to 52 zg/ml with a mean of 21.3. All levels were in the effec-
CHLORAMPHENICOL
METABOLISM
tive therapeutic levels, namely above 10 ig/ ml. Hence the dose of 25 mg/kg was a suitable one to use for therapeutic effectiveness. Five children were studied a second time after undergoing nutritional rehabilitation for 4-8 weeks. During this period, their body weights had improved by 8-10%. Figures 3 and 4 show the responses of plasma values initially and after treatment, the normal curve is shown for comparison. Three obvious findings are: 1) Peak levels were achieved later than normal in children with PCM. The interval decreased with nutritional improvement. 2) Peak levels were initially high but became lower after rehabilitation. 3) Drug was cleared slowly at first; this improved after
excretion
979
40
-
NORMAL
30
3 YEARS
MALE
TIME
IN
HOURS
4
30
-
AP
3 YEARS
F WITH
-
PCM
GRADE
III
P(M
/TT:I:G1DPM
S TIME
FIG.
4.
2 IN
24
levels after oral body weight.
500
E__]
400
I
f]
in PCM
3’.
HOURS
Plasma chloramphenicol of drug-25 mg drug/kg
administration
rehabilitation. It was also apparent that the abnormal pattern started to improve while the recovery in body weight was not yet complete. Urinary
IN CHILDREN
UNCHANGED CONJUGATED
a,
Detailed information was available for five children with PCM before and after 4-8 weeks of rehabilitation. Patterns of these patients were similar. One of these is presented in the Fig. 5. Initially the drug could be recovered in samples collected at 30 hours in as high a concentration as 100 zg/ml. Only 35-55% of the drug was present in the conjugated form and the remainder was excreted unchanged in the urine. Rehabilitation of the child was associated with a significant change in the excretory pattern. Much higher concentrations were achieved during the initial period,
40
i30 \
z
20 $0
HALF LIFE 53 HR
U 24
30
4O 3O GRADE
2O U 4
.-‘GRADE
10
III PCM IIPCM
3 HR
4
4I?
4
12 TIME
IN
REHASILITATED 24
30
HOURS
FIG. 3. Plasma chloramphenicol levels after oral administration of drug-25 mg drug/kg body weight.
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5. Excretion in urine
m
GRADE
ratio of conjugated in a malnourished
U
and child.
free
chlor-
i.e., 0-8 hours. Very small quantities could be recovered beyond 12 hours. This pattern is akin to the normal. Following rehabilitation, conjugated drug constituted 55-65% of the excreted total. The component was still less than that excreted by the normal child. The change of excretory pattern became evident even while the somatic recovery was only 8-10% in body weight. Liver biopsy was carried out in two children with grade II malnutrition and 7-8 mg of tissue were obtained. The activity of bilirubin-UDP transferase was 0.14 and 0.24 mg bilirubin conjugated/g of liver tissue. The values in normal adults from the same laboratory range between 0.45 to 0.95 mg/g of tissue. The values for normal children have not been established.
980
MEHTA
Discussion Observation in the normal infants showed that highest plasma levels of chloramphenicol was reached in 2 hours and it fell to a trace level by 12 hours. The pattern was very similar to that observed by Kelly and coworkers (3) in children who were given 22 mg/kg single dose and it also resembled the one observed in adults administered a 2-g single dose (10). Hence chloramphenicol palmitate in a fine suspension was easily absorbed and the pattern of decay was similar to that seen in adults receiving crystalline chloramphenicol. The children suffering from PCM showed three significant features in the plasma curve in response to oral administration of the drug. 1) Peak plasma levels were generally attained in 4 hours or more in contrast to 2 hours in the normal. 2) The peak plasma levels were generally higher in children suffering from PCM than in the controls. 3) The drug took much longer to clear from the plasma of malnourished children. Furthermore all three features tended to revert to a normal pattern in the five children who were restudied after nutritional rehabilitation. The longer time taken to achieve the peak level may be due to slower absorption. Sixty to eighty percent of children suffering from PCM of the severity studied by us show evidence of inability to absorb nutrients such as fats, carbohydrates and vitamin B52 (2). Abnormalities of the mucosa of the small bowel in the form of villous atrophy have been well described (2, 8). Under such conditions it is conceivable that chloramphenicol is not absorbed rapidly. The peak level of drug in plasma, however, is higher in malnourished infants. This cannot be explained on the basis of defective absorption. As drug absorption begins, so do the processes of distribution, binding to plasma proteins, biotransformation and excretion. The plasma level reflects the total influence of all processes in combination. Disturbances in any of these processes could result in higher plasma levels in PCM. In infants with PCM, it took longer for the drug to clear from blood. An interference
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ET AL. with distribution or protein binding cannot be explained easily by both of these phenomena occurring simultaneously. In normal adults, the drug is bound to plasma albumin to the extent of6O%. In PCM plasma albumin levels fall. The bound form should therefore fall also. Under such circumstances the plasma clearance should not be delayed. However, we have no information at present on the exact nature and extent ofprotein binding in PCM. Delayed clearance of the drug as well as the higher peak levels in plasma can result from either a slower biotransformation or a slower renal clearance. Studies on renal function in PCM (personal communication, 0. P. Ghai) did not reveal any significant change either in glomerular filtration rate or in tubular reabsorption. The unchanged chloramphenicol is chiefly dependent on glomerular filtration and conjugated form on tubular secretion for elimination (4). Renal excretory mechanism may not be responsible for the behaviour of plasma levels. This area needs more work for complete elucidation. In PCM changes in liver cells have long been documented. In addition to gross histologic changes, electron-microscopic changes have been described in the endoplasmic reticulum in humans (12) as well as in experimental animals (6, 7). Most microsomal enzymes involved in metabolism of drugs reside in the endoplasmic reticulum. It is therefore possible that alteration in biotransformation of chloramphenicol by the hepatocyte may be responsible for the behaviour of the plasma curve. In two children, liver biopsies could be obtained before the drug was administered. Glucuronyl
transferase
was
estimated
using
bilirubin as substrate (13). The liver tissue demonstrated a reduction of the enzyme to one-half and one-third, respectively, of the normal value. Although such information gathered is preliminary, bilirubin glucuronyl transferase is probably similar to the enzyme needed for chloramphenicol conjugation. It can be postulated that a similar diminution of specific enzyme might interfere with an efficient conjugation of the drug. The data obtained from urine analysis support this postulate. Three patients with PCM excreted a large amount of unchanged drug (50-60%) in the
CHLORANIPHENICOL
METABOLISM
urine. When these children were restudied after initial rehabilitation a greater amount of conjugated drug was excreted. The pattern in the normal child showed that 75-85% of the drug in the urine was in the conjugated form. In adults nearly 90% of the drug is excreted in the conjugated form (10). The observations of the present study point to the need for a reevaluation of therapy in malnourished children. When using chloramphenicol a possibly slower rate of metabolism results in higher blood levels which take a longer time to clear. The current practice of drug administration in dosages calculated only on the basis of body weight are likely to be hazardous in a malnourished child. A reduction in dosage and wider spacing of administration may be required. Exact recommendations must be based on more extended observations and a study of multiple dose administration. Summary Metabolism of orally administered chloramphenicol palmitate was studied in ten malnourished children and four normal children between the ages of 6 months and 3 years. Drug was given in a single dose of 25 mg/kg body weight and blood samples and timed urine samples collected at 1, 2, 4, 8, 12, 24 and 30 hours. Drug was analyzed in the serum and urine. In urine the total as well as unconjugated drug was estimated. The malnourished children achieved a peak level which was delayed but higher than the drug from the normal. In the urine they excreted larger amounts of unchanged drug than the normal child. After nutritional rehabilitation these findings resembled those in normal children. The observations are explained on the basis of a slower rate of conjugation. Bilirubin-UDP transferase activity was estimated in two malnourished children and
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981
IN CHILDREN
found to be lower than evidence lends support slow biotransformation.
in normal adults. to the hypothesis
This of
El
References 1. LEVINE, J., AND H. FISCUBACK. The chemical determination of chloramphenicol in biological materials. Antibiotics I: 59, 1951. 2. GUPTE S. P., S. NIEHTA AND B. N. S. WALIA. Small bowel function in protein-calorie malnutrition. Indian Pediat. 7:481-488, 1970. 3. KELLY R. S., A. D. HUNT AND S. G. TASHMAN. Studies on the absorption and distribution of chloramphenicol. Pediatrics 8: 362-367, 1951. 4. WEINSTEIN, L. Pharmacological Basis of Therapeutics (4th ed), edited by Goodman L. S. and A. Gilman. New York: Macmillan, 1970. 5. DELIWALA, K. E. V., AND U. K. SHETH. Chloramphenicol levels in the blood following oral administration of various chloramphenicol esters. J. Postgraduate Med., Bombay 8: 27-32, 1962. 6. OROY J. M., T. SAMORAJSKI, R. R. ZIMMERMAN ANt) P. NI. RADY. Effects of postnatal protein deficiency on weight gain, serum, proteins enzymes cholesterol and liver ultrastructure in a subhuman primate. Am. J. Pathol. 48: 796, 1966. 7. Svooi,s D., G. GRADY AND J. I-IIGGINSON. The effects of chronic protein deficiency in rats. II. Biochemical and ultrastructure changes. Lab. Invest. 15: 731, 1966. 8. BRUNSER 0., W. RElu, F. NIONCKERERG, A. MACCIONI, I. CONTRERAS AND E. TRABUCCO. Jejunal 38: 605, 9.
GLAZKO
E.
L.
biopsies 1966.
in
A. J., A. W. An
infant KINKEL,
malnutrition.
W. C.
Pediatrics ALEGNANI
ANt)
evaluation of the absorption characteristics of different chloramphenicol preparation in normal human subjects. Clin. Pharmacol. Therap. 9: 472, 1968. 10. GLAZKO A. J. Biochemical studies on chloramphenicol. II. Tissue distribution and excretion studies. J. Pharmacol. Exptl. Therap. 96: 445-459, 1949. II. AGERIAR, A. J., A. W. KINKEL AND J. C. SAMYN. Effects of polymorphism on the absorption of chloramphenicol from chloramphenicol palmitate. J. Pathol. Sci. 56: 847 -853, 1967. 12. THERON, J. J., ANt) N. LIENBERG. Some observations on fine cytology of parenchymal liver cells in kwashiorkor. J. Pathol. Bacteriol. 86: 109, 1963. 13. BLACK, NI. AND B. BILLING. Determination of bilirubin UDP transferase activity in human liver biopsies. Clin. Chem. Acta 29: 27. 1970. HOLMES.
