Complement protein-calorie Robert M.D.,
activity in children malnutrition”2
Suskind,3 Anong
M.D.,
Pariyanonda,5
M.Sc.,
ABSTRACT system
Using
on hospital 50 it was children
hemolytic severe
and
recovery.
The
in contrast with
the CH,0
serum
(P
is compromised ence
of
AC
untreated
PCM
activity
assay,
malnutrition
in children
(7%)
results and
provides
serum indicate
that J. Clin.
Children with protein-calorie malnutrition are known to be unusually susceptible to severe infection (1, 2). PCM causes depression of several host defense mechanisms, including phagocytic and killing functions of leukocytes (3), cell-mediated immune response (4, 5), inflammatory response (5), and antibody production (6). Recent reports have also suggested that PCM adversely affects a child’s complement system (4, 6, 7). In order to evaluate the functional integrity of the complement system, we studied the total hemolytic complement (C H 50) activity before, during, and after treatment of malnourished Thai children. In addition, we looked for possible anticomplementary (AC) activity in serum of the same patients.
(PCM)
its
(PCM)
with
Kulapongs,
serum
that,
greater
repair Nutr.
for
29:
actmvity
inversely
coincides lO89
hospital
was significantly
with
the healthy
activity during
in their later
to the amount
with
PCM,
intake
depressed 1092,
than
(AC)
AC
in children
mechanism
complement their
less
On day 8 it rose to normal, and by day CH5, titer of 16 well-nourished febrile
significantly
correlated
the
during
kwashiorkor
anticomplementary
had detectable
a possible Am.
PCM,
evidenced
in a PCM
These
children.
(CH,0)
calorie
of untreated
two
functionally, activity
CH,0
11(40%)
titer
< 0.01).
complement protein
in 17 healthy control subjects. than the controls. The mean
or I or 4, but only
Significantly, that
mean
to that
PCM,
Panja
we evaluated
the
was,
USAR,
MC,
Ph.D.
with
Of the children
LTC,
and Stitaya Sirisinha,’ D.D.S.,
children
days I and 4 than significantly higher
day
M.D.,
28
of
admission
either
Edelman,4
Robert
with
controls. serum
of AC
activity
the complement
of adequate complement
on
convalescence.
diet. activity
in
system The
pres-
in some
1976.
kwashiorkor (MK), and 14 had kwashiorkor diagnoses were based on the scoring system et al. (8). The children were treated with
(K); these of McLaren intravenous
fluid, broad spectrum antibiotics, supplemental vitamins and minerals, and gradually increasing calorie and protein intakes. By the end of the first week, the patients were receiving daily doses of 100 kcal, I g of protein/kg; by day 29 the dosage had increased to 175 kcal, 4 g of protein/kg per day. Most patients were studied on days I (admission), 4, 8, 29, 50, and 71 for CH5, and AC activity. Seventeen healthy northern Thai village children, ages I to 5, and eight clinically well-nourished children admitted to Bangkok Children’s Hospital with febrile illnesses comprised the two control patient groups. The eight febrile patients included six with fevers of unknown From
the
Anemia
and
Malnutrition
Research
Cen-
Twenty-eight children, ages I to 5 years, were admitted to the research ward of the Anemia and Malnutrition Research Center mn Chiang Mai, Thailand. The clinical and laboratory examinations determined that the majority of PCM children had bacterial infections involving the blood, skin, and urinary, gastrointestinal, and respiratory tracts. Several children had fevers of unknown origin; others had septic shock on admission or during the first week. On admission four children were clinically
ter, Chiang Mai University/St. Louis University School of Medicine, Chiang Mai Thailand, and the Department of Biochemistry and Pediatrics, St. Louis University School of Medicine, St. Louis, Missouri. 2 Supported by National Institutes of Health Grant no. 11044, United States Army Medical Research and Development Command, and the Rockefeller Foundation. Address all correspondence: Dr. Robert Suskind, Associate Professor of Pediatrics and Clinical Nutrition, Associate Program Director, Clinical Research Center (El 8-479), Massachusetts Institute of Technology, 50 Ames Street, Cambridge, Massachusetts 02139. Department of Virology, SEATO Medical Research Laboratory, Bangkok, Thailand. ‘Department of Mi-
classified
crobiology,
Methods
The
as having
American
Journal
marasmus
of Clinical
(M),
10 had
Nutrition
marasmus-
29: OCTOBER
Downloaded from https://academic.oup.com/ajcn/article-abstract/29/10/1089/4617090 by Cornell University Library user on 11 June 2018
1976,
Mahidol
pp.
1089-1092.
University,
Printed
Bangkok,
in U.S.A.
Thailand.
1089
SUSKIND
1090 origin. one with bus meningitis.
typhoid This
ET
AL.
fever, and another with tubercugroup received blood tests upon
admission to determine CH,0 and AC activity. Serum was stored at -70 C for no more than I month before assay. We used a standard assay of total CH50 activity (9) to determine its activity in the serum. Briefly, sheep erythrocytes were sensitized with anti-sheep erythrocyte immunoglobulin (hemolysin). The sensitized erythrocytes were then added to serial 2-fold dilutions of the patient’s serum, mixed, and incubated at 37 C and 4 C. The results of this assay are expressed as the reciprocal of the serum dilution (units/mi) producing 50% hemolysis of the sensitized sheep erythrocytes. In the AC activity assay the patient’s serum was mixed in equal volume with a standard serum of known CH,0 titer. The mixture was then incubated at 37 C for 30 mm, and the total hemolytic titer of the mixture was determined as previously described (9). For dilution control, heat-inactivated standard serum (56 C for 30 mm)
and
saline
were
each
mixed
in equal
volumes
the unheated standard serum (37 C for 37 mm), CH,0 titer of the mixture then determined.
the
Because the admission and subsequent titers of the M and MK groups did not differ significantly, they were combined into one group for purposes of analysis and referred to as the M-MK group. Comparing the mean CH50 titers of the children with M-MK to that of the healthy control children, we found no significant difference CH50
the
day
1, 4,
and
8 values
of
the
two
groups (Fig. 1). With nutritional repair, however, the M-MK values rose significantly above the control on days 29 (P < 0.001) and 50 (P < 0.01). Upon this group’s discharge, I
M
-
MEAN
± SE
P,#{128}ANI
MK
K
8OO-
600
400 I
200
(no HethyFev I Control Control
4
FIG. I. Comparable in the healthy control admission and during groups.
8
29
50
7
HOSPITAL
I
4
8
29
DAYS
with
and
Results
among
HOSPITAL
FIG. 2. Percentage of children with M-M K or with K who had detectable serum AC activity. Numbers over the bars represent the number of patients tested.
50
71
DAYS
serum CH,, titers on admission and fever control groups, and on convalescence in the M-MK and K
Downloaded from https://academic.oup.com/ajcn/article-abstract/29/10/1089/4617090 by Cornell University Library user on 11 June 2018
the difference between its mean CH50 value and that of the control was no longer significant. The mean CH50 titer of the children with K was lower on admission than that of the M-MK group. It was also significantly less than the control values on both day 1 (P < 0.05) and day 4 (P < 0.01). The titers rose, however, after day 4, and were significantly greater than the controls by day 50. On day 71 they fell to near control levels. Of the eight fever control patients, the mean CH50 was significantly higher than the mean of the 17 healthy controls. The mean CH50 titer of the children with M-MK and K was significantly lower than the febrile controls on days I and 4. When sera from 17 healthy and eight fever control children were each mixed with a standard serum of 960 to 1,280 units/mI, the resulting CH50 activity titer was always greater than 480 units/mI. Thus, a PCM serum that reduced CH50 activity of the standard serum to 320 units/ml or less indicated the presence of AC activity in that PCM serum. Two (14%) of the M-MK group and five (36%) of the K group evidenced AC activity in their serum on day 1; on day 4, 21% of the M-MK group and 31% of the K group had AC activity in their serum (Fig. 2). A total of four children (29%) from the
M-MK
group
and seven (50%)
from
the K
group showed evidence of AC activity on days I and/or 4. The percent of children with serum AC activity decreased after day 4, and
COMPLETE
ACTIVITY
IN
none showed evidence of activity on day 50 or 71. Figure 3 illustrates the relationship between the CH50 titer and the functional level of AC activity in repeated sera drawn from a child that died of sepsis on hospital day 33. Figure 4 shows that, the lower the CH50 titer in a PCM serum on day 1, the greater will be the reduction of the CH50 titer in a control serum mixed with that PCM serum. Thus, a significant reciprocal correlation exists between the CH50 titer of a PCM serum and the amount of detectable AC activity in that serum (P < 0.01).
CHILDREN
WITH
Recent investigators have noted that an impaired complement system in children with PCM may account, in part, for the increased susceptibility of malnourished children to bacterial infection (4, 6, 7). Smythe et al. (4) were the first to call attention to altered CH50 activity, while Chandra (6) described depressed C3 concentrations in children with PCM. Sirisinha et al. (7), in perhaps the most extensive study to date of the complement system in PCM, found that the serum concentration of Clq, Cls, C3, CS, C6, C8, C9, and C3 proactivator were all depressed in children with untreated PCM (7). Our study confirms the functional impairment of the complement system in children with K and, in addition, relates increased CH50 activity to the improved clinical status of the patients studied. Finally, the results demonstrate the prestoo 90
#{149}-.
CH5
x-x
ANTICOMPLEMENTARY
100
TITER TITER
z
80
I
>0 Zc o
Qo
I #{176} 60 -J
0
z 3
5#{176} 40
30
-) -1 33
2
tO HOSPITAL
20
30
DAYS
FIG. 3. CH,0 and AC activity in sequential child with kwashiorkor who ultimately died whelming sepsis on hospital day 33.
sera of a from over-
Downloaded from https://academic.oup.com/ajcn/article-abstract/29/10/1089/4617090 by Cornell University Library user on 11 June 2018
1091
I40O I200
1000 2
800E
600
400’ I.I-.
o
200
2 C)
0 200 CH50
Discussion
PCM
TITER
600 (urits/mI)
000 IN
CONTROL’
14.00 PCM
SERA
FIG. 4. Correlation coefficient between Cl-I,0 titers in admission PCM sera and in a control serum mixed 1:1. with each admission PCM sera (AC activity assay).
ence of serum AC activity with depressed CH50 titers. The complement system potentiates opsonification, immune adherence, phagocytosis, and white cell chemotaxis (10). Although CH50 activity in PCM is low, no study to date has shown that opsonizing complement activity in PCM serum is similarly impaired (11, 12). The mechanisms leading to depressed CH50 activity in PCM children are under study. Because the well-nourished patients with fever showed higher CH50 titers than the healthy control (Fig. I), it is unlikely that the fever that occurred in many PCM children lowered their CH50 activity. Sirisinha et al. (7) provide evidence that a child’s nutritional state is probably the major factor causing depressed complement protein concentrations. The presence of AC activity in the serum of these children and the rapid rise and fall of CH,0 activity over a period of days (Fig. 3) indicate that complement consumption should also be considered a factor responsible for the depressed complement factors in some children with PCM. Alper et al. (13) reported lowered CH50 activity and isolated depression of C3 concentrations in a patient. However, the measured concentration of C3 was considerably lower than that noted in our series of patients (7). The elevated CH50 activity levels on days 29 and 50 (Fig. I) may express functionally the “rebound” elevation of serum complement
SUSKIND
1092
protein concentrations previously observed during convalescence of malnourished Thai children (7). The AC activity detected in 40% of the patients on hospital days I and 4, together with the correlation between presence of AC activity and depression of Ch50, suggest that AC activity may contribute to lowered CH50 activity in the serum of some PCM patients. Several substances have been associated with AC activity; these include: circulating immune complexes (14), endotoxin (15), macroglobulins (16), CI-esterase inhibitor (17), and C3 and C6 inhibitors (18). The present study does not identify which of these factors may be operative, but recent work by Oberle et al. (19) indicates that a large percentage of children with PCM have evidence of circulating endotoxin.
a
ET
AL.
6. CHANDRA, R. K. Immunocompetence in undernutrition. J. Pediat. 81: 1 194, 1972. 7. SIRISINHA, S., R. SUSKIND, R. EDEI,MAN, C. CHARUPATANA AND R. E. OLSON. Complement and C3 proactivator levels in children with protein-calorie malnutrition and effect of dietary treatment. Lancet 8.
9.
10.
11.
12.
I: 1016, 1973. MCLAREN, D. S., R. L. PELLETT ANt) W. W. C. READ. A simple scoring system for classifying the severe forms of protein-calorie malnutrition of early childhood. Lancet I: 533, 1967. KABAT, E. A., AND M. M. MAYER. Complement and complement fixation: experimental immunochemistry. SprinfieId, Illinois: Charles C Thomas, 1961, p. 133. GIGLI, 1., ANt) K. F. AUSTEN. Phylogeny and function of the complement system. Ann. Rev. Microbiol. 25: 309, 1971. SETH, V., AND R. K. CHANDRA. Opsonic activity, phagocytosis and bactericidal capacity of polymorphs in undernutrition. Arch. Diseases Childhood 47: 282, 1972. TEJADA, C., V. ARGUETA, M. SANCHEZ AND C.
Phagocytic and alkaline phosphatase activity of leukocytes in kwashiorkor. J. Pediat. 64: 753, 1964. 13. ALPER, C. A., K. J. BLOCH ANt) F. S. ROSEN. Increased susceptibility to infection in a patient with Type II essential hypercatabolism of C3. New EngI. J. Med. 288: 601, 1973. 14. PICKERING, R. J., H. GEWLRZ AND R. A. GooD. Complement inactivation by serum from patients with acute and hypocomplementemic chronic gbALBERTAZZI.
The authors are indebted to Khun Tawat Tositarat and Robert Parker for their outstanding contribution toward the completion of this study. We also acknowledge the excellent editorial assistance of Susanna Fein and Judith Katz-Levine.
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