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Cell mediated immune responses in children with iron deficiency and combined iron and zinc deficiency
NUTRITION RESEARCH, Vol. 8, pp. 129-136, 1988 0271-5317/88 $3.00 + .00 Printed in the USA. Copyright (c) 1988 Pergamon Journals Ltd. All rights reserved.
C E L L M E D I A T E D IMMUNE R E S P O N S E S IN C H I L D R E N W I T H IRON AND C O M B I N E D IRON A N D ZINC D E F I C I E N C Y *
A.
Sabri
Kemahl~,
Department
M.D.,
of Pediatrics,
DEFICIENCY
Emel Babacan, M.D., A y h a n O. Qavdar, M.D. M e d i c a l School of A n k a r a University, Ankara, T u r k e y * *
ABSTRACT This study has been c a r r i e d out to e v a l u a t e cellm e d i a t e d immune r e s p o n s e in c h i l d r e n w i t h iron d e f i c i e n c y alone and iron d e f i c i e n c y a s s o c i a t e d W i t h zinc deficiency, comparatively. T h i r t y p a t i e n t s w e r e studied, w i t h t h i r t e e n having iron and zinc deficiency. A b s o l u t e l y m p h o c y t e counts were found to be above 1 2 0 0 / c u m m whereas, E - r o s e t t e forming T - L y m p h o c y t e s w e r e s i g n i f i c a n t l y lower than the control subjects in b o t h groups, w i t h m u c h lower v a l u e s for c o m b i n e d iron and zinc d e f i c i ~ h t children. A l t h o u g h delayed h y p e r s e n s i t i v i t y r e s p o n s e s w e r e i m p a i r e d in b o t h groups of patients, it was found to be m u c h l o w e r in c o m b i n e d ironzinc deficient group. It is c o n c l u d e d t h a t b o t h iron d e f i c i e n c y and i r o n - a n d zinc deficiency m a y impair cell m e d i a t e d immune responses, h o w e v e r z i n c m a y have a g r e a t e r influence. KEY WORDS
: Anemia,
cellular
iron deficiency, immunity.
zinc d e f i c i e n c y ,
INTRODUCTION Iron d e f i c i e n c y anemia is the m o s t c o m m o n n u t r i t i o n a l d e f i c i e n c y in children and is w i d e s p r e a d t h r o u g h o u t the world(l). Its frequency varies among c o m m u n i t i e s ; h o w e v e r the p r o b l e m of i n a d e q u a t e and/or i m b a l a n c e d nutrition, t o g e t h e r with the high i n c i d e n c e of p a r a s i t i c d i s e a s e s and g a s t r o i n t e s t i n a l b l o o d loss, c e n t r i b u t e s to the d e v e l o p m e n t of n e g a t i v e iron b a l a n c e and iron d e f i c i e n c y anemia. * This study was supported, in part, by Turkish Scientific and Technical Research Council (TUBITAK). ** Address correspondence and reprint request to: Prof.Dr.Ayhan O.~avdar, Cinnah Caddesi, 62/12, ~ankaya-Ankara, TURKEY.
129
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A.S. KEMAHLI et al.
I n c r e a s e d s u s c e p t i b i l i t y to infections has been o b s e r v e d in iron d e f i c i e n t p e o p l e and this has led to the i n v e s t i g a t i o n s of immune status of t h e s e patients. In studies c o n c e r n i n g humoral i m m u n i t y serum i m m u n o g l o b u l i n s , complement C~ levels and antibody r e s p o n s e s to v a r i o u s a n t i g e n s w e r e found to De normal (2,3). As of cellular immunity T - l y m p h o c y t e values (E-rosette), absolute l y m p h o c y t e counts (ALC), l y m p h o b l a s t i c t r a n s f o r m a t i o n in response to v a r i o u s m i t o g e n s and in v i v o delayed cutaneous h y p e r s e n t i v i t y (DCH) r e a c t i o n s h a v e been studied. A l t h o u g h most authors have found a d e c r e a s e in c e l l - m e d i a t e d i m m u n e responses, there are some who have found no d i f f e r e n c e c o m p a r e d with the normal p o p u l a t i o n (3-6) On the o t h e r h a n d zinc is k n o w n to have i m p o r t a n t roles in m a n y systems and functions, i n c l u d i n g the immune system. Thus, immune status of zinc d e f i c i e n t subjects have also b e e n studied. E s p e c i a l l y i m p a i r m e n t of c e l l - m e d i a t e d immunity has been shown by d e c r e a s e s of T - l y m p h o c y t e s , l y m p h o b l a s t i c t r a n s f o r m a t i o n rates, a b s o l u t e l y m p h o c y t e c o u n t s a n d impairment of d e l a y e d c u t a n e o u s hypersensitivitiy (7-15). A l t h o u g h i m m u n o l o g i c a l status in iron and zinc d e f i c i e n c i e s have been studied by v a r i o u s investigators, zinc levels of patients w i t h iron d e f i c i e n c y have not b e e n reported, p a r t i c u l a r l y in p u b l i c a t i o n s r e g a r d i n g iron d e f i c i e n c y and immunity. On the other hand, effects of t h e s e two d e f i c i e n c i e s on immune f u n c t i o n s have not been studied c o m p a r a t i v e l y . Therefore, this study has been u n d e r t a k e n to d e t e r m i n e w h i c h trace element's d e f i c i e n c y is r e s p o n s i b l e of it, and if b o t h are effective, w h e t h e r there is a q u a n t i t a t i v e d i f f e r e n c e b e t w e e n the two.
MATERIALS
AND METHODS
This study c o n s i s t e d of 30 patients (10 boys, 20 girls) b e t w e e n the ages of 7 m o n t h s and 20 y e a r s (Mean age 9.5 y e a r s ) . T h e y had initial d i a g n o s i s of iron d e f i c i e n c y anemia, and w e r e studied in P e d i a t r i c H e m a t o l o g y D i v i s i o n and Zinc Research Unzt. Care was taken to assure that the c h i l d r e n studied did not have any apparent h e a l t h problems; like infections, protein and/or c a l o r i e deficiency, etc., e x c e p t their anemias. Serum a l b u m i n was above 3.4 g/dl in each case. In all p a t i e n t s serum iron, total iron b i n d i n g c a p a c i t y and serum or p l a s m a zinc levels were d e t e r m i n e d and 13 p a t i e n t s h a v e b e e n f o u n d w i t h zinc d e f i c i e n c y in addition to iron deficiency. Thus, 17 P a t i e n t s with pure iron d e f i c i e n c y and 13 w i t h c o m b i n e d iron and zinc deficiency were d i v i d e d in two groups. Ten h e a l t h y c h i l d r e n w i t h similar ages c o m p r i s e d the control group. H e i g h t s and w e i g h t s of pure iron d e f i c i e n t children were b e t w e e n 1 0 t h - 5 0 t h c e n t i l e s for age, Whereas the m e a s u r e m e n t s of the c o m b i n e d i r o n - z i n c d e f i c i e n t children were b e l o w 5th c e n t i l e for age, on N C H S (National Center for Health Statistics) g r o w t h charts. The a n t h r o p o m e t r i c m e a s u r e m e n t s of the control group c h i l d r e n w e r e b e t w e e n 2 5 t h - 9 5 t h centiles for age. Written p a r e n t a l c o n s e n t was o b t a i n e d b e f o r e entry into this study. The d i a g n o s i s of iron d e f i c i e n c y a n e m i a was established in accordance w i t h the r e c c o m m e n d a t i o n s of WHO, i.e., hemoglobin b e l o w i0 g/dl, serum iron b e l o w 69 ~g/dl and t r a n s f e r r i n s a t u r a t i o n below 15 % (16). Patients w e r e c o n s i d e r e d zinc deficient if their
CELLULAR IMMUNITY, IRON AND ZINC
131
p l a s m a zinc levels w e r e below 83 ~g/dl, which was 2 s t a n d a r d d e v i a t i o n s b e l o w o u r laboratory normal value of 106 ~g/dl. S e r u m iron, total iron b i n d i n g c a p a c i t y (TIBC) and serum zinc l e v e l s have b e e n d e t e r m i n e d by previously d e s c r i b e d m e t h o d s ( 1 7 ) . (Zinc w a s m e a s u r e d by atomic a b s o r p t i o n s p e c t r o p h o t o m e t e r , PerkinE l m e r m o d e l 103). A b s o l u t e L y m p h o c y t e Count (ALC), T - l y m p h o c y t e counts (E-rosette assay), and skin tests of p u r i f i e d p r o t e i n d e r i v e d antigen (PPD) a n d d i n i t r o c h o r o b e n z e n e (DNCB) were u s e d to assess c e l l - m e d i a t e d immunity. ALC w e r e d e r i v e d from the c o m p l e t e blood c o u n t a n d d i f f e r e n t i a l count. T h e p e r c e n t a g e of l y m p h o c y t e s in p e r i p h e r a l b l o o d smear was m u l t i p l i e d by the white blood cell c o u n t s as folows: % lymphocyte XWBC Absolute lymphocyte count= i00 T - l y m p h o c y t e count (E-rosette) m e t h o d o f J o n d a l et al (18).
was o b t a i n e d
by the m o d i f i e d
T u b e r c u l i n (PPD) skin test was e v a l u a t e d only in c h i l d r e n who were BCG v a c c i n a s in the last five years. RT 23 T W 80 solution, c o n t a i n i n g 5TU/10.1 ml was used and an i n d u r a t i o n of 5 m m or m o r e Was c o n s i d e r e d a p o s i t i v e response. E r y t h e m a W a s not c o n s i d e r e d as a c r i t e r i o n for p o s i t i v e response. Dinitrochlorobenzene (l-chloro-2, 4-dinitrobenzene) was a p p l i e d and e v a l u a t e d as d e s c r i b e d by C a t a l o n e et al. (19). A s e n s ~ i z i n g dose of 2000 ~g a n d a c h a l l a n g e dose of 50 ~g were used. S t a t i s t i c a l Analyses: L o g a r i t h m i c t r a n s f o r m a t i o n Was a p p l i e d to A L C and inverse sinus (angle value; sin-l.x) t r a n s f o r m a t i o n for E - r o s e t t e values. T h e p r e s e n c e of s t a t i s t i c a l l y d i f f e r e n t groups w a s d e t e r m i n e d by v a r i a n c e analysis and ~he d e t e r m i n a t i o n of d i f f e r e n t g r o u p s by Duncan test for ALC, E-rosettes. Skin tests w e r e a n a l y s e d by Z-test (for proportions) (20).
RESULTS M e a n s e r u m iron levels of pure iron d e f i c i e n t and c o m b i n e d i r o n - z i n c d e f i c i e n t children w e r e 33.9• ~g/dl and 29.1• ~g/dl (x• r e s p e c t i v e l y . Mean t r a n s f e r r i n saturation of p u r e iron d e f i c i e n t group was 5.8• %, and the c o m b i n e d i r o n - z i n c d e f i c i e n t g r o u p had a m e a n t r a n s f e r r i n s a t u r a t i o n of 5.0• % P u r e iron d e f i c i e n t c h i l d r e n h a d a m e a n serum zinc level of 1 1 0 . 9 • ~g/dl, w h e r e a s c h i l d r e n w i t h c o m b i n e d iron and zinc d e f i c i e n c y had a m e a n value of 68.7• ~ g / d l . T h e s e values for control subjects w e r e 98• ~g/dl for serum iron, 35 % for t r a n s f e r r i n s a t u r a t i o n (range 25-50 %) and 9 9 . 7 • ~g/dl for serum zinc, respectively. M e a n ALC w e r e 1786 ; i84/cu m m (X~ SX) in pure iron d e f i c i e n t subjects, 2515 • 4 0 4 / c u mr~ in iron and zinc d e f i c i e n t patients, 3i~1 • 3~5/c~ nun.in control subjects. %'here was a s i g n i f i c a n t d e c r e a s e in iron d e f i c i e n c y group compared with the c o n t r o l group (Table i).
132
A.S. KEMAHLI et al. T A B L E
Absolute
Lymphocyte
~ron Deficiency
Iron-Zinc Deficiency
15 1786 la4
n S~
I !
1
C o u n t s (ALC) In P a t i e n t A n d C o n t r o l and The Statistical Analysis
Control
13 2515 404
p > 0.05.
J! p
Groups
10 3181 385
p>0.05
! |
Mean E-Rosette values were 34.05• % in i r o n d e f i c i e n t cases, 2 5 . 4 4 • 1 . 5 2 % in c o m b i n e d i r o n - z i n c d e f i c i e n t g r o u p and 44.25 • 1.61% in c o n t r o l c a s e s . T h e r e w e r e s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s b e t w e e n a l l g r o u p s (Table 2).
E-Rosette
n R SR S i n -I x
Values
T A B L E @ in P a t i e n t a n d Control
Iron Deficiency
Iron-Zinc Deficiency
i0 34.05 2.93 37.29 ! L
ll 25.44 1.52 30~19 IJ < 0.05-
P < 0.01 ~ p
Groups
Control i0 44.25 1.61 41.69 p<0.01
-
P P D W a S e v a l u a t e d o n l y in B C G v a c c i n a t e d c a s e s f i v e years. A p o s i t i v e r e s u l t w a s o b s e r v e d in 8 3 . 3 i r o n d e f i c i e n t c a s e s , in 62.5 % (5/8) of i r o n z l n c a n d 80 % (8/10) of c o n t r o l s .
! J in the l a s t % (5/7) of deficient cases
S i m i l a r l y , p o s i t i v e r e s p o n s e s to D N C B w e r e o b s e r g e d in 35.3 % (6/i7) of p u r e i r o n d e f i c i e n c y p a t i e n t s , in 7.7 % (1/13) of Cohlbined i r o n - z i n c d e f i c i e n c y p a t i e n t s , & n d in 90 % (9/10) of c o n t r o l s . P o s i t i v e s k i n t e s t s w e r e o b t a i n e d in ii o u t o f 23 t e s t s in i r o n p l u s z i n c d e f i c i e n t c a s e s a n d in 17 o u t of 20 s k i n t e s t s in 10 c o n t r o l c h i l d r e n . S t a t i s t i c a l l y t h e r e w a s no s l g n i f i c a n t d i f f e r e n c e b e t w e e n the two p a t i e n t g r o u p s , w h e r e a s h h e r e w e r e s i g n i f i c a n t d i f f e r e n c e s b e t w e e n the c o n t r o l g r o u p a n d the two p a t i e n t g r o u p s (Table 3). When DNCB results were considered alone there Were s i g n i f i c a n t d i f f e r e n c e s b e t w e e n a l l t h r e e g r o u p s (Table
4).
P P D r e s u l t s w e r e n o t a n a l y s e d s e p a r a t e l y d u e to the s m a l l n u m b e r of cases. B o t h s k i n tests w e r e p o s i t i v e in 50 % (3/6) of p ~ r e i r o n d e f i c i e n c y , in 0 % (0/8) of c o m b i n e d i r o n a n d z i n c a e f i c i e n c y g r o u p s a n d 70 % (7/10) o f c o n t r o l s . On the o t h e r h a n d b o t h skin t e s t s w e r e n e g a t i v e in 16 % (1/6) o f p u r e i r o n d e f i c i e n c y c a s e s , in 3 7 . 5 % (3/8) of i r o n a n d z i n c d e f i c i e n c y c a s e s a n d 0 % (0/i0) of c o n t r o l s .
CELLULAR IMMUNITY, IRON AND ZINC TABLE Delayed
Cutaneous
3
Hypersensitivity
Responses
Iron Deficiency No. of skin tests Positive responses P e r c e n t a g e (%)
23 ii 4~8
21 6 28.6
p < 0.05
No.
Cutaneous
of cases
Positive responses P e r c e n t a g e (%)
Iron Deficiency 17 6
!L--p<0.0Z
4 Responses
to DNCB
Control i0 9
7.7
p <0.01
P1 P < o.o1-
!
j
Iron-Zinc Deficiency 13 1
35.3
I,,
Control 20 17 85.0
p< o.o1-
TABLE Hypersensitivity
I
To PPD and DNCB
Iron-Zinc Deficiency
I
Delayed
133
90.9
p< 0.01
I
_I
DISCUSSION A l t h o u g h m o s t aspects of c e l l h l a r immune r e s p o n s e s have b e e n explored, still there are some d i s c r e p a n c i e s u n r e s o l v e d in the l i t e r a t u r e (3-6). The e f f e c t s of iron and zinc d e f i c i e n c i e s on cell m e d i a t e d immunity, have b e e n i n v e s t i g a t e d c o m p a r a t i v e l y , in this ss As it was shown previoulsy, mean ALC of both p a t i e n t groups w e r e above 1200 c e l l s / c u mm, w h i c h is c o n s i d e r e d the c r i t i c a l level for i y m p h o p e n i a (21). In p r e v i o u s reports, ALC of iron d e f i c i e n t p a t i e n t s have b e e n found to be normal and to rise after iron t h e r a p y (22, 23). It has been shown that zinc d e p r i v a t i o n a f f e c t s the d e v e l o p m e n t of l y m p h o i d t i s s u e s , e s p e c i a l l y thymus (7, 12, 24). Thus it can be e x p e c t e d that zinc d e f i c i e n c e y may a f f e c t T - l y m p h o c y t e s , as well. A l t h O u g h lymphopenia is r e p o r t e d in p a t i e n t s with liver cirrhosis, w h i c h is an example of conditional zinc d e f i c i e n c y , and in some animals made zinc deficient, no s i g n i f i c a n t d i f f e r e n c e was o b s e r v e d in leucocyte counts of old p e o p l e by the a d d i t i o n of zinc to their diets(10,15). T-ceil immune f u n c t i o n s and E - r o s e t t e values are known to be a f f e c t e d a d v e r s e l y in iron d e f i c i e n c y and it has been shown to rise after iron t h e r a p y by v a r i o u s i n v e s t i g a t o r s (2, 5, 8, 9, 22, 25). It has b e e n d e m o n s t r a t e d that zinc is n e c e s s a r y for T - c e l l functions a n d the d r o p in E - r o s e t t e v a l u e s is an index of the d r o p in s e r u m t h y m i c h o r m o n e levels (8, 13, 26). The addition of zinc to diets i n c r e a s e d E - r o s e t t e forming T - c e l l s c o n s i d e r a b l y in e l d e r e l y individuals (10). The findings in our study show that T-cell f u n c t i o n s are a f f e c t e d from the d e f i c i e n c y of both trace elements, the i n f l u e n c e of zinc being greater.
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Many authors have investigated skin reactivity to various antigens in iron deficiency and have found considerable decreases, compared with the controls (2-6,22). Some have also found conversion to positive skin response after therapy (5,22). The proposed mechanism is impaired DNA synthesis and a decrease in T-cells(2,22). Delayed cutaneous hypersensitivity in zinc deficiency has been investigated in patients with malnutrition, acrodermatitis enteropathica, prolonged parenteral therapy, elderly people, some immune deficiency syndromes, some malignancies, hematologic disorders, infectious diseases and Down syndrome. By zinc supl~entation orally, or even topically to skin the hyporeactivity observed in zinc deficiency states returned to normal (7, i0,ii, 13,]4, 27, 28). We have f o ~ d a si/3nificant decrease in delayed hypersensitivity reactions among iron deficient and iron-zinc deficient patients. Although the reactions of iron-zinc deficiency group were lower than pure iron deficient ones, there was no statistically significant difference between the two groups. However, when responses to DNCB were considered alone, difference became significant between two groups. As a conclusion both iron deficiency and combined iron-zinc deficiency may impair cell-mediated immune response, the latter having greater influence. REFERENCES I. Lanzkowsky P. Iron metabolism and iron deficiency anemia. In Miller, DR. Pearson, HA, Baehner, RL, McMillan, CW, eds. Smith's Blood Diseases of Infancy and Childhood, 4th edition. Saint Louis: The CV Mosby Company, 1978: 108. 2. Chandra RK, Saraya AK.I~paired immunocompetence associated with iron deficiencey. J. Pediatr. 1975; 86: 899-902. 3. McDougall LG, Anderson R, MacNab GM, Katz J. The immune response in iron- deficient children: Impaired cellular defense mechanisms with altered humoral components. J.Pediatr. 1975; 86: 833-43. 4. Joynson D ~ , Jacobs A, Walker DM, Dolby AE. Defect of cellmediated immunity in patients With iron deficiency anemia. Lancet 1972; ii: 1058 - 9. 5. Krantmann HJ, Young SR, Ank BJ, O'Donnell CM, Rachelefsky GS, Stiehm ER. Immune function in pure iron deficiency. Am.J.Dis. Child. 1982; 136:840-4. 6. Kulapongs P, Vithayasai V, Suskind R, Olson RE. Cell-mediated immunity and phagocytosis and killing function in children with severe iron deficiency anemia. Lancet 1974; ii: 689-91. 7. Bach JF. The multi-faceted zinc dependency of immune system. In: Seligman M, Hitzig WH, ed. INSERM symposium No: 16. Elsevier North Holland, 1980: 225-7. 8. Chandra RK. Cell-mediated immunity in nutritional Proc. 1980; 39: 3088-92.
imbalance.
Fed.
9. Chandra RK, Vyas D, Chandra S. Trace element regulation of immunity and infection. In: Abstracts, International Symposium on the Health Effects and Interactions of Essential and Toxic Elements, Lund, Sweden, 1983: 38.
CELLULAR IMMUNITY, IRON AND ZINC 10.
135
Duchateau J, Delepesse G, Vriens R, Collet H. Beneficial effects of oral zinc supplementation on the immune response of old people. Am.J.Med. 1981; 70:1001-4.
ll. Golden ~ N , Golden BA, Harland PSEG, Jackson AA. Zinc and immunocopetence in protein-energy malnutrition. Lancet 1978;1 1226-7 . 12. Golden MHN, Jackson AA, Golden BE. Effects of zinc on thymus of recently malnourished children. Lancet 1977; ii: 1057-9. 13. Good RA. Zinc and immunity. In: Abstracts, International Symposium on the Health Effects and Interactions of Essential and Toxic Elements. Lund, Sweden, 1983: 37. 14. Pekarek RS, Sandstead HH, Jacob RA, Barcome DF. Abnormal cellular immune responses during acquired zinc deficiency. Am.J.Clin.Nutr. 1979; 32: 1466-71. 15. Prasad AS. Clinical, biochemical and pharmacological role of zinc Ann. Rev. Phormacol. Toxicol. 1979; 20:393-426. 16. World Health organization. Geneva, 1972: No. 503.
Nutritional
anemias.
Tech. Rep. Ser.
17. Qavdar AO, Arcasoy A, Cin $. Babacan E, G~zda@o~l~ S.Geophagia in Turkey: Iron and zinc deficiency, iron and zinc absorption studies and response to treatment with zinc in geophagia cases. In: Prasad AS, ~avdar AS, Brewer GJ, Aggett PJ eds. Zinc Deficiency in Human Subjects, New York: Alan R. Liss, Inc., 1983: 71-97. 18. Jondal H, Hom G, Wigzell H. Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming non immune rosettes with sheep red blood cells. J.Exp. Med. 19~2; 136: 207-15. 19. Catalone WJ, Taylor P, Rabson AS, Chretien PB. A method for dinitrochlorobenzene contact sensetization. N.Engl.J.Med. 1972; 286: 399-402. 20. Sokal RR, Rohlf FJ, Biometry. Francisco; 1969.
WH Freeman
and Comp.
2nd ed.
San
21. Ammann AJ, Fudenberg HH. Immunodeficiency states. In: Fudenberg HH, Sites DF, Caldwell JL, Wells JV, ed. Basic and Clinical Immunology. 3rd ed. Lange Medical publications, Los Altos, California, 1980: 420. 22. Bhaskaram deficient
C, Reddy V. Cell-mediated children. Br.Med.J. 1975;
immunity 3:522.
in iron-and-vitamin
23. Fletcher J, Mather J, Lewis MJ, Whiting G.Mouth Lesions in iron deficiency anemia: relationship to Candida albicans in salvia and to impairment of lympocyte transformation. J.Infect. Dis. 1975; 131:44-50.
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24. Beach RS, Gershwin ME, Makishima RK, Hurley LS. Impaired immunologic ontogeny in postantal zinc deprivation. J.Nutr. 1980; 110: 805-15. 25. Srikantia SG, Prasad JS, Bhaskaram C, Krishnamachari KAVR- Anemia and the immune response. Lancet; 1976; i: 1307-9. 26. Zanzonico P, Fernandes G, Good RA. The differential sensitivitiy of T-cell and B-cell mitogenesis to in vitro zinc deficiency. Cell. Immunol. 1981; 60: 203-11. 27. Chandra RK. Acrodermatitis enteropathica: zinc levels and cell-mediated immunity. Pediatrics 1980; 66: 789-91. 28. Allen JI, Kay NE, McClain CJ. Severe zinc deficiency in humans: association with a reversible T-lymphocyte dysfuntion. Ann. Intern. Med. 1981; 95: 154-7.
Accepted.for publication August 21, 1987
C E L L M E D I A T E D IMMUNE R E S P O N S E S IN C H I L D R E N W I T H IRON AND C O M B I N E D IRON A N D ZINC D E F I C I E N C Y *
A.
Sabri
Kemahl~,
Department
M.D.,
of Pediatrics,
DEFICIENCY
Emel Babacan, M.D., A y h a n O. Qavdar, M.D. M e d i c a l School of A n k a r a University, Ankara, T u r k e y * *
ABSTRACT This study has been c a r r i e d out to e v a l u a t e cellm e d i a t e d immune r e s p o n s e in c h i l d r e n w i t h iron d e f i c i e n c y alone and iron d e f i c i e n c y a s s o c i a t e d W i t h zinc deficiency, comparatively. T h i r t y p a t i e n t s w e r e studied, w i t h t h i r t e e n having iron and zinc deficiency. A b s o l u t e l y m p h o c y t e counts were found to be above 1 2 0 0 / c u m m whereas, E - r o s e t t e forming T - L y m p h o c y t e s w e r e s i g n i f i c a n t l y lower than the control subjects in b o t h groups, w i t h m u c h lower v a l u e s for c o m b i n e d iron and zinc d e f i c i ~ h t children. A l t h o u g h delayed h y p e r s e n s i t i v i t y r e s p o n s e s w e r e i m p a i r e d in b o t h groups of patients, it was found to be m u c h l o w e r in c o m b i n e d ironzinc deficient group. It is c o n c l u d e d t h a t b o t h iron d e f i c i e n c y and i r o n - a n d zinc deficiency m a y impair cell m e d i a t e d immune responses, h o w e v e r z i n c m a y have a g r e a t e r influence. KEY WORDS
: Anemia,
cellular
iron deficiency, immunity.
zinc d e f i c i e n c y ,
INTRODUCTION Iron d e f i c i e n c y anemia is the m o s t c o m m o n n u t r i t i o n a l d e f i c i e n c y in children and is w i d e s p r e a d t h r o u g h o u t the world(l). Its frequency varies among c o m m u n i t i e s ; h o w e v e r the p r o b l e m of i n a d e q u a t e and/or i m b a l a n c e d nutrition, t o g e t h e r with the high i n c i d e n c e of p a r a s i t i c d i s e a s e s and g a s t r o i n t e s t i n a l b l o o d loss, c e n t r i b u t e s to the d e v e l o p m e n t of n e g a t i v e iron b a l a n c e and iron d e f i c i e n c y anemia. * This study was supported, in part, by Turkish Scientific and Technical Research Council (TUBITAK). ** Address correspondence and reprint request to: Prof.Dr.Ayhan O.~avdar, Cinnah Caddesi, 62/12, ~ankaya-Ankara, TURKEY.
129
130
A.S. KEMAHLI et al.
I n c r e a s e d s u s c e p t i b i l i t y to infections has been o b s e r v e d in iron d e f i c i e n t p e o p l e and this has led to the i n v e s t i g a t i o n s of immune status of t h e s e patients. In studies c o n c e r n i n g humoral i m m u n i t y serum i m m u n o g l o b u l i n s , complement C~ levels and antibody r e s p o n s e s to v a r i o u s a n t i g e n s w e r e found to De normal (2,3). As of cellular immunity T - l y m p h o c y t e values (E-rosette), absolute l y m p h o c y t e counts (ALC), l y m p h o b l a s t i c t r a n s f o r m a t i o n in response to v a r i o u s m i t o g e n s and in v i v o delayed cutaneous h y p e r s e n t i v i t y (DCH) r e a c t i o n s h a v e been studied. A l t h o u g h most authors have found a d e c r e a s e in c e l l - m e d i a t e d i m m u n e responses, there are some who have found no d i f f e r e n c e c o m p a r e d with the normal p o p u l a t i o n (3-6) On the o t h e r h a n d zinc is k n o w n to have i m p o r t a n t roles in m a n y systems and functions, i n c l u d i n g the immune system. Thus, immune status of zinc d e f i c i e n t subjects have also b e e n studied. E s p e c i a l l y i m p a i r m e n t of c e l l - m e d i a t e d immunity has been shown by d e c r e a s e s of T - l y m p h o c y t e s , l y m p h o b l a s t i c t r a n s f o r m a t i o n rates, a b s o l u t e l y m p h o c y t e c o u n t s a n d impairment of d e l a y e d c u t a n e o u s hypersensitivitiy (7-15). A l t h o u g h i m m u n o l o g i c a l status in iron and zinc d e f i c i e n c i e s have been studied by v a r i o u s investigators, zinc levels of patients w i t h iron d e f i c i e n c y have not b e e n reported, p a r t i c u l a r l y in p u b l i c a t i o n s r e g a r d i n g iron d e f i c i e n c y and immunity. On the other hand, effects of t h e s e two d e f i c i e n c i e s on immune f u n c t i o n s have not been studied c o m p a r a t i v e l y . Therefore, this study has been u n d e r t a k e n to d e t e r m i n e w h i c h trace element's d e f i c i e n c y is r e s p o n s i b l e of it, and if b o t h are effective, w h e t h e r there is a q u a n t i t a t i v e d i f f e r e n c e b e t w e e n the two.
MATERIALS
AND METHODS
This study c o n s i s t e d of 30 patients (10 boys, 20 girls) b e t w e e n the ages of 7 m o n t h s and 20 y e a r s (Mean age 9.5 y e a r s ) . T h e y had initial d i a g n o s i s of iron d e f i c i e n c y anemia, and w e r e studied in P e d i a t r i c H e m a t o l o g y D i v i s i o n and Zinc Research Unzt. Care was taken to assure that the c h i l d r e n studied did not have any apparent h e a l t h problems; like infections, protein and/or c a l o r i e deficiency, etc., e x c e p t their anemias. Serum a l b u m i n was above 3.4 g/dl in each case. In all p a t i e n t s serum iron, total iron b i n d i n g c a p a c i t y and serum or p l a s m a zinc levels were d e t e r m i n e d and 13 p a t i e n t s h a v e b e e n f o u n d w i t h zinc d e f i c i e n c y in addition to iron deficiency. Thus, 17 P a t i e n t s with pure iron d e f i c i e n c y and 13 w i t h c o m b i n e d iron and zinc deficiency were d i v i d e d in two groups. Ten h e a l t h y c h i l d r e n w i t h similar ages c o m p r i s e d the control group. H e i g h t s and w e i g h t s of pure iron d e f i c i e n t children were b e t w e e n 1 0 t h - 5 0 t h c e n t i l e s for age, Whereas the m e a s u r e m e n t s of the c o m b i n e d i r o n - z i n c d e f i c i e n t children were b e l o w 5th c e n t i l e for age, on N C H S (National Center for Health Statistics) g r o w t h charts. The a n t h r o p o m e t r i c m e a s u r e m e n t s of the control group c h i l d r e n w e r e b e t w e e n 2 5 t h - 9 5 t h centiles for age. Written p a r e n t a l c o n s e n t was o b t a i n e d b e f o r e entry into this study. The d i a g n o s i s of iron d e f i c i e n c y a n e m i a was established in accordance w i t h the r e c c o m m e n d a t i o n s of WHO, i.e., hemoglobin b e l o w i0 g/dl, serum iron b e l o w 69 ~g/dl and t r a n s f e r r i n s a t u r a t i o n below 15 % (16). Patients w e r e c o n s i d e r e d zinc deficient if their
CELLULAR IMMUNITY, IRON AND ZINC
131
p l a s m a zinc levels w e r e below 83 ~g/dl, which was 2 s t a n d a r d d e v i a t i o n s b e l o w o u r laboratory normal value of 106 ~g/dl. S e r u m iron, total iron b i n d i n g c a p a c i t y (TIBC) and serum zinc l e v e l s have b e e n d e t e r m i n e d by previously d e s c r i b e d m e t h o d s ( 1 7 ) . (Zinc w a s m e a s u r e d by atomic a b s o r p t i o n s p e c t r o p h o t o m e t e r , PerkinE l m e r m o d e l 103). A b s o l u t e L y m p h o c y t e Count (ALC), T - l y m p h o c y t e counts (E-rosette assay), and skin tests of p u r i f i e d p r o t e i n d e r i v e d antigen (PPD) a n d d i n i t r o c h o r o b e n z e n e (DNCB) were u s e d to assess c e l l - m e d i a t e d immunity. ALC w e r e d e r i v e d from the c o m p l e t e blood c o u n t a n d d i f f e r e n t i a l count. T h e p e r c e n t a g e of l y m p h o c y t e s in p e r i p h e r a l b l o o d smear was m u l t i p l i e d by the white blood cell c o u n t s as folows: % lymphocyte XWBC Absolute lymphocyte count= i00 T - l y m p h o c y t e count (E-rosette) m e t h o d o f J o n d a l et al (18).
was o b t a i n e d
by the m o d i f i e d
T u b e r c u l i n (PPD) skin test was e v a l u a t e d only in c h i l d r e n who were BCG v a c c i n a s in the last five years. RT 23 T W 80 solution, c o n t a i n i n g 5TU/10.1 ml was used and an i n d u r a t i o n of 5 m m or m o r e Was c o n s i d e r e d a p o s i t i v e response. E r y t h e m a W a s not c o n s i d e r e d as a c r i t e r i o n for p o s i t i v e response. Dinitrochlorobenzene (l-chloro-2, 4-dinitrobenzene) was a p p l i e d and e v a l u a t e d as d e s c r i b e d by C a t a l o n e et al. (19). A s e n s ~ i z i n g dose of 2000 ~g a n d a c h a l l a n g e dose of 50 ~g were used. S t a t i s t i c a l Analyses: L o g a r i t h m i c t r a n s f o r m a t i o n Was a p p l i e d to A L C and inverse sinus (angle value; sin-l.x) t r a n s f o r m a t i o n for E - r o s e t t e values. T h e p r e s e n c e of s t a t i s t i c a l l y d i f f e r e n t groups w a s d e t e r m i n e d by v a r i a n c e analysis and ~he d e t e r m i n a t i o n of d i f f e r e n t g r o u p s by Duncan test for ALC, E-rosettes. Skin tests w e r e a n a l y s e d by Z-test (for proportions) (20).
RESULTS M e a n s e r u m iron levels of pure iron d e f i c i e n t and c o m b i n e d i r o n - z i n c d e f i c i e n t children w e r e 33.9• ~g/dl and 29.1• ~g/dl (x• r e s p e c t i v e l y . Mean t r a n s f e r r i n saturation of p u r e iron d e f i c i e n t group was 5.8• %, and the c o m b i n e d i r o n - z i n c d e f i c i e n t g r o u p had a m e a n t r a n s f e r r i n s a t u r a t i o n of 5.0• % P u r e iron d e f i c i e n t c h i l d r e n h a d a m e a n serum zinc level of 1 1 0 . 9 • ~g/dl, w h e r e a s c h i l d r e n w i t h c o m b i n e d iron and zinc d e f i c i e n c y had a m e a n value of 68.7• ~ g / d l . T h e s e values for control subjects w e r e 98• ~g/dl for serum iron, 35 % for t r a n s f e r r i n s a t u r a t i o n (range 25-50 %) and 9 9 . 7 • ~g/dl for serum zinc, respectively. M e a n ALC w e r e 1786 ; i84/cu m m (X~ SX) in pure iron d e f i c i e n t subjects, 2515 • 4 0 4 / c u mr~ in iron and zinc d e f i c i e n t patients, 3i~1 • 3~5/c~ nun.in control subjects. %'here was a s i g n i f i c a n t d e c r e a s e in iron d e f i c i e n c y group compared with the c o n t r o l group (Table i).
132
A.S. KEMAHLI et al. T A B L E
Absolute
Lymphocyte
~ron Deficiency
Iron-Zinc Deficiency
15 1786 la4
n S~
I !
1
C o u n t s (ALC) In P a t i e n t A n d C o n t r o l and The Statistical Analysis
Control
13 2515 404
p > 0.05.
J! p
Groups
10 3181 385
p>0.05
! |
Mean E-Rosette values were 34.05• % in i r o n d e f i c i e n t cases, 2 5 . 4 4 • 1 . 5 2 % in c o m b i n e d i r o n - z i n c d e f i c i e n t g r o u p and 44.25 • 1.61% in c o n t r o l c a s e s . T h e r e w e r e s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s b e t w e e n a l l g r o u p s (Table 2).
E-Rosette
n R SR S i n -I x
Values
T A B L E @ in P a t i e n t a n d Control
Iron Deficiency
Iron-Zinc Deficiency
i0 34.05 2.93 37.29 ! L
ll 25.44 1.52 30~19 IJ < 0.05-
P < 0.01 ~ p
Groups
Control i0 44.25 1.61 41.69 p<0.01
-
P P D W a S e v a l u a t e d o n l y in B C G v a c c i n a t e d c a s e s f i v e years. A p o s i t i v e r e s u l t w a s o b s e r v e d in 8 3 . 3 i r o n d e f i c i e n t c a s e s , in 62.5 % (5/8) of i r o n z l n c a n d 80 % (8/10) of c o n t r o l s .
! J in the l a s t % (5/7) of deficient cases
S i m i l a r l y , p o s i t i v e r e s p o n s e s to D N C B w e r e o b s e r g e d in 35.3 % (6/i7) of p u r e i r o n d e f i c i e n c y p a t i e n t s , in 7.7 % (1/13) of Cohlbined i r o n - z i n c d e f i c i e n c y p a t i e n t s , & n d in 90 % (9/10) of c o n t r o l s . P o s i t i v e s k i n t e s t s w e r e o b t a i n e d in ii o u t o f 23 t e s t s in i r o n p l u s z i n c d e f i c i e n t c a s e s a n d in 17 o u t of 20 s k i n t e s t s in 10 c o n t r o l c h i l d r e n . S t a t i s t i c a l l y t h e r e w a s no s l g n i f i c a n t d i f f e r e n c e b e t w e e n the two p a t i e n t g r o u p s , w h e r e a s h h e r e w e r e s i g n i f i c a n t d i f f e r e n c e s b e t w e e n the c o n t r o l g r o u p a n d the two p a t i e n t g r o u p s (Table 3). When DNCB results were considered alone there Were s i g n i f i c a n t d i f f e r e n c e s b e t w e e n a l l t h r e e g r o u p s (Table
4).
P P D r e s u l t s w e r e n o t a n a l y s e d s e p a r a t e l y d u e to the s m a l l n u m b e r of cases. B o t h s k i n tests w e r e p o s i t i v e in 50 % (3/6) of p ~ r e i r o n d e f i c i e n c y , in 0 % (0/8) of c o m b i n e d i r o n a n d z i n c a e f i c i e n c y g r o u p s a n d 70 % (7/10) o f c o n t r o l s . On the o t h e r h a n d b o t h skin t e s t s w e r e n e g a t i v e in 16 % (1/6) o f p u r e i r o n d e f i c i e n c y c a s e s , in 3 7 . 5 % (3/8) of i r o n a n d z i n c d e f i c i e n c y c a s e s a n d 0 % (0/i0) of c o n t r o l s .
CELLULAR IMMUNITY, IRON AND ZINC TABLE Delayed
Cutaneous
3
Hypersensitivity
Responses
Iron Deficiency No. of skin tests Positive responses P e r c e n t a g e (%)
23 ii 4~8
21 6 28.6
p < 0.05
No.
Cutaneous
of cases
Positive responses P e r c e n t a g e (%)
Iron Deficiency 17 6
!L--p<0.0Z
4 Responses
to DNCB
Control i0 9
7.7
p <0.01
P1 P < o.o1-
!
j
Iron-Zinc Deficiency 13 1
35.3
I,,
Control 20 17 85.0
p< o.o1-
TABLE Hypersensitivity
I
To PPD and DNCB
Iron-Zinc Deficiency
I
Delayed
133
90.9
p< 0.01
I
_I
DISCUSSION A l t h o u g h m o s t aspects of c e l l h l a r immune r e s p o n s e s have b e e n explored, still there are some d i s c r e p a n c i e s u n r e s o l v e d in the l i t e r a t u r e (3-6). The e f f e c t s of iron and zinc d e f i c i e n c i e s on cell m e d i a t e d immunity, have b e e n i n v e s t i g a t e d c o m p a r a t i v e l y , in this ss As it was shown previoulsy, mean ALC of both p a t i e n t groups w e r e above 1200 c e l l s / c u mm, w h i c h is c o n s i d e r e d the c r i t i c a l level for i y m p h o p e n i a (21). In p r e v i o u s reports, ALC of iron d e f i c i e n t p a t i e n t s have b e e n found to be normal and to rise after iron t h e r a p y (22, 23). It has been shown that zinc d e p r i v a t i o n a f f e c t s the d e v e l o p m e n t of l y m p h o i d t i s s u e s , e s p e c i a l l y thymus (7, 12, 24). Thus it can be e x p e c t e d that zinc d e f i c i e n c e y may a f f e c t T - l y m p h o c y t e s , as well. A l t h O u g h lymphopenia is r e p o r t e d in p a t i e n t s with liver cirrhosis, w h i c h is an example of conditional zinc d e f i c i e n c y , and in some animals made zinc deficient, no s i g n i f i c a n t d i f f e r e n c e was o b s e r v e d in leucocyte counts of old p e o p l e by the a d d i t i o n of zinc to their diets(10,15). T-ceil immune f u n c t i o n s and E - r o s e t t e values are known to be a f f e c t e d a d v e r s e l y in iron d e f i c i e n c y and it has been shown to rise after iron t h e r a p y by v a r i o u s i n v e s t i g a t o r s (2, 5, 8, 9, 22, 25). It has b e e n d e m o n s t r a t e d that zinc is n e c e s s a r y for T - c e l l functions a n d the d r o p in E - r o s e t t e v a l u e s is an index of the d r o p in s e r u m t h y m i c h o r m o n e levels (8, 13, 26). The addition of zinc to diets i n c r e a s e d E - r o s e t t e forming T - c e l l s c o n s i d e r a b l y in e l d e r e l y individuals (10). The findings in our study show that T-cell f u n c t i o n s are a f f e c t e d from the d e f i c i e n c y of both trace elements, the i n f l u e n c e of zinc being greater.
134
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Many authors have investigated skin reactivity to various antigens in iron deficiency and have found considerable decreases, compared with the controls (2-6,22). Some have also found conversion to positive skin response after therapy (5,22). The proposed mechanism is impaired DNA synthesis and a decrease in T-cells(2,22). Delayed cutaneous hypersensitivity in zinc deficiency has been investigated in patients with malnutrition, acrodermatitis enteropathica, prolonged parenteral therapy, elderly people, some immune deficiency syndromes, some malignancies, hematologic disorders, infectious diseases and Down syndrome. By zinc supl~entation orally, or even topically to skin the hyporeactivity observed in zinc deficiency states returned to normal (7, i0,ii, 13,]4, 27, 28). We have f o ~ d a si/3nificant decrease in delayed hypersensitivity reactions among iron deficient and iron-zinc deficient patients. Although the reactions of iron-zinc deficiency group were lower than pure iron deficient ones, there was no statistically significant difference between the two groups. However, when responses to DNCB were considered alone, difference became significant between two groups. As a conclusion both iron deficiency and combined iron-zinc deficiency may impair cell-mediated immune response, the latter having greater influence. REFERENCES I. Lanzkowsky P. Iron metabolism and iron deficiency anemia. In Miller, DR. Pearson, HA, Baehner, RL, McMillan, CW, eds. Smith's Blood Diseases of Infancy and Childhood, 4th edition. Saint Louis: The CV Mosby Company, 1978: 108. 2. Chandra RK, Saraya AK.I~paired immunocompetence associated with iron deficiencey. J. Pediatr. 1975; 86: 899-902. 3. McDougall LG, Anderson R, MacNab GM, Katz J. The immune response in iron- deficient children: Impaired cellular defense mechanisms with altered humoral components. J.Pediatr. 1975; 86: 833-43. 4. Joynson D ~ , Jacobs A, Walker DM, Dolby AE. Defect of cellmediated immunity in patients With iron deficiency anemia. Lancet 1972; ii: 1058 - 9. 5. Krantmann HJ, Young SR, Ank BJ, O'Donnell CM, Rachelefsky GS, Stiehm ER. Immune function in pure iron deficiency. Am.J.Dis. Child. 1982; 136:840-4. 6. Kulapongs P, Vithayasai V, Suskind R, Olson RE. Cell-mediated immunity and phagocytosis and killing function in children with severe iron deficiency anemia. Lancet 1974; ii: 689-91. 7. Bach JF. The multi-faceted zinc dependency of immune system. In: Seligman M, Hitzig WH, ed. INSERM symposium No: 16. Elsevier North Holland, 1980: 225-7. 8. Chandra RK. Cell-mediated immunity in nutritional Proc. 1980; 39: 3088-92.
imbalance.
Fed.
9. Chandra RK, Vyas D, Chandra S. Trace element regulation of immunity and infection. In: Abstracts, International Symposium on the Health Effects and Interactions of Essential and Toxic Elements, Lund, Sweden, 1983: 38.
CELLULAR IMMUNITY, IRON AND ZINC 10.
135
Duchateau J, Delepesse G, Vriens R, Collet H. Beneficial effects of oral zinc supplementation on the immune response of old people. Am.J.Med. 1981; 70:1001-4.
ll. Golden ~ N , Golden BA, Harland PSEG, Jackson AA. Zinc and immunocopetence in protein-energy malnutrition. Lancet 1978;1 1226-7 . 12. Golden MHN, Jackson AA, Golden BE. Effects of zinc on thymus of recently malnourished children. Lancet 1977; ii: 1057-9. 13. Good RA. Zinc and immunity. In: Abstracts, International Symposium on the Health Effects and Interactions of Essential and Toxic Elements. Lund, Sweden, 1983: 37. 14. Pekarek RS, Sandstead HH, Jacob RA, Barcome DF. Abnormal cellular immune responses during acquired zinc deficiency. Am.J.Clin.Nutr. 1979; 32: 1466-71. 15. Prasad AS. Clinical, biochemical and pharmacological role of zinc Ann. Rev. Phormacol. Toxicol. 1979; 20:393-426. 16. World Health organization. Geneva, 1972: No. 503.
Nutritional
anemias.
Tech. Rep. Ser.
17. Qavdar AO, Arcasoy A, Cin $. Babacan E, G~zda@o~l~ S.Geophagia in Turkey: Iron and zinc deficiency, iron and zinc absorption studies and response to treatment with zinc in geophagia cases. In: Prasad AS, ~avdar AS, Brewer GJ, Aggett PJ eds. Zinc Deficiency in Human Subjects, New York: Alan R. Liss, Inc., 1983: 71-97. 18. Jondal H, Hom G, Wigzell H. Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming non immune rosettes with sheep red blood cells. J.Exp. Med. 19~2; 136: 207-15. 19. Catalone WJ, Taylor P, Rabson AS, Chretien PB. A method for dinitrochlorobenzene contact sensetization. N.Engl.J.Med. 1972; 286: 399-402. 20. Sokal RR, Rohlf FJ, Biometry. Francisco; 1969.
WH Freeman
and Comp.
2nd ed.
San
21. Ammann AJ, Fudenberg HH. Immunodeficiency states. In: Fudenberg HH, Sites DF, Caldwell JL, Wells JV, ed. Basic and Clinical Immunology. 3rd ed. Lange Medical publications, Los Altos, California, 1980: 420. 22. Bhaskaram deficient
C, Reddy V. Cell-mediated children. Br.Med.J. 1975;
immunity 3:522.
in iron-and-vitamin
23. Fletcher J, Mather J, Lewis MJ, Whiting G.Mouth Lesions in iron deficiency anemia: relationship to Candida albicans in salvia and to impairment of lympocyte transformation. J.Infect. Dis. 1975; 131:44-50.
136
A.S. KEMAHLIet al.
24. Beach RS, Gershwin ME, Makishima RK, Hurley LS. Impaired immunologic ontogeny in postantal zinc deprivation. J.Nutr. 1980; 110: 805-15. 25. Srikantia SG, Prasad JS, Bhaskaram C, Krishnamachari KAVR- Anemia and the immune response. Lancet; 1976; i: 1307-9. 26. Zanzonico P, Fernandes G, Good RA. The differential sensitivitiy of T-cell and B-cell mitogenesis to in vitro zinc deficiency. Cell. Immunol. 1981; 60: 203-11. 27. Chandra RK. Acrodermatitis enteropathica: zinc levels and cell-mediated immunity. Pediatrics 1980; 66: 789-91. 28. Allen JI, Kay NE, McClain CJ. Severe zinc deficiency in humans: association with a reversible T-lymphocyte dysfuntion. Ann. Intern. Med. 1981; 95: 154-7.
Accepted.for publication August 21, 1987