Superoxide dismutase activity and blastogenic response of lymphocytes from copper-deficient rats fed diets containing fructose or cornstarch

NUTRITION RESEARCH, Vol. 9, pp. 273-282, 1989 0271-5317/89 $3.00 + .00 Printed in the USA. Copyright (c) 1989 Pergamon Press plc. All rights reserved.

SUPEROXIDE D ~ E ACTIVITY AND BIASIDG~2~C RESPONSE OF LYMPHOC~TES FROM C O P P E R - D E F I ~ RATS FED DIETS F~3~ OR CORNSTARf~ Uma Babu, M.S. and Mark L. Failla, Ph.D. 1

Vitamin and Mineral Nutrition laboratory, USDA, A~/riculture Research Service, Beltsville, Maryland 20705 and Graduate Program in Nutritional Sciences, University of Maryland, College Park, Maryland 20742 ABSTRACT Male weanling rats were pair-fed diets containing either fructose (F) or starch (S) and either 7 ppm (+) or 0.7 ppm (-) copper (Cu) for 4-5 weeks. The activity of Cu, Zn-superoxide dismutase in splenocytes, cervical l y ~ node cells (CLNC) and thymocytes and the copper content of thymocytes were lower in rats fed -Cu diets than +Cu diets. Moreover, these indicators of Cu status were lower when rats were fed -Cu diets with F than with S. T-lymphocyte function was assessed by m~a~uring mitogen-stimulated E~A synthesis in cultures of lymphoid cells ~ t e d in medium containing 2% sera pooled from rats fed either the identical diet (homologous system) or the diet with same carbohydrate but different level of Cu (heterologous system) as the tissue donor. In the homologous system, mitogen-irnhlced DNA synthesis was lower in splenocytes, higher in CI/~C, and unchanged in thymocytes isolated from rats fed -Cu diets than in the respective cells from +Cu rats. Mitogenic reactivity was inhibited to a greater degree in splenocytes from rats fed -Cu diets containing F instead of S. There was a tendency fo~ unstinrdated cells from -Cu rats to incorporate ~ g h e r levels of H-thymidine than cells from oopper-adequate rats. ~H-thymidine inoorporation in unstimulated cells from rats fed -O1 diets tended to be significantly different in cultures containing heterologous sera than those in cultures containing homologous sera. In contrast, source of sera did not affect mitogen-induced ONA synthesis. These results d~nonstrate that the i~pact of dietary copper deficiency on the function of T-lymphocytes is organ specific and can be influenced by the type of dietary carbohydrate. Key words:

Copper deficiency, lymphocytes, T-cells, mitogens, dietary carbohydrate

1 Address all correspondence to Dr. Mark L. Failla, USDA, ARS, ~qRC, VMNL, Bldg. 307, Rm. 205, BARC-East, Beltsville, Maryland 20705

273

274

U. BABU and M. FAILLA

It has been well established that adeq0a~te levels of dietary copper are essential for the function of the immune system (1-3). We recently found that the hi, oral ~ response of young male rats fed copper-deficient diets was markedly influenced by the type of dietary carbohydrate (4). More specifically, rats fed a copper-deficient diet with stard~ had a significantly higher titer of antiboc[ies to ~ e ~ after primary and ~__~zor~a_ ~y immunization than rats fed a copper-deficient diet containing fructose. Studies have been initiated to examine the effects of the dietary ocpper-carbohydrate interaction on the function of the different classes of immune oells, viz., T and B lymphocytes and m a ~ . In the studies stm~_a__~izedbelow, we investigated the effects of dietary stalr~ and fructose on the co,per status of lymphoid c e n s a ~ the mi~genic responsiveness of T - l y m ~ isolated f.uw spleen, t h y ~ m and cervical lymph nodes of rats fed diets with a~Tuate and deficient levels of copper. The influence of sera obtained from rats fed copper-adequate and copper-deficient diets on the activity of l y ~ in vitro was also. MATERIAIS AND ~ Animals. Male Lewis rats (N=28) were obtained as weanlings (40-50g) from Charles River Breeding Laboratories (Wilmingtm~, MA) and housed separately in stainless steel c~.~es with wire-meshed bottums in a room with c=mroiled tem~ex-ature (21-22 C), humidity (45-50%) and light/dark cycle (lights on 0600-1800 h). Distilled deionized water was provided ad libitum. Diets. The basal diet contained the following c c ~ per kg: 627.3g fructose (F) or starch (S); 200g egg white p,Dtein; 95g corn oil; 30g cellulose; 35g AIN-76 mineral mix (5) without cupric carbonate; 10g AIN-76A vitamin mix (5); 2.7g choline bitartarate; and, O.O02g biotin. These diets conta~ 0.6-0.75mg Cu/kg as determined by atomic absorption s p e c t r o s c ~ (AAS) and are referred to as copper-deficient (-Cu). Copper sufficient (+Cu) diets were prepared by supplementing the mineral mix with cupric carbonate so that the diet contained 6.7-7.1rag C~/kg. Experimental Desiqn. All animals were fed a diet containing starch and adequate copper (S+Cu) for 2 days after arrival. Then rats were divided into four groups and fed one of the following diets: fructose with adequate copper (F+Cu) ; fructose with deficient copper (F-Cu) ; starch with adequate copper (S+Cu) ; and starch with deficient copper (S-Oa). The amount of food given each day to animals fed F+Cu, S+Cu and S-O/ diets was equal to the average amount by a g e - m a ~ rats fed F-O/ diet. Food intake by rats fed F-Cu diet was measured twice each week and all aDimals were weighed weekly. On days 28-35, one rat from each treatment group was weighed, anesthetized, and blood was obtained by cardiac puncture. A separate aliquot was collected in a heparinized capillary tube after severing the vena cava to determine hematccrit. Lymphoid tissues were weighed and placed in ice-cold RPMI 1640 medium. Isolation and culture of 17mphocytes. Cell suspensions ware prepared by teasing cervical lymph nodes and thymus with sterile stainless steel needles and by pressing pieoes of spleen between sterile glass s l J ~ . Aliquc~s (7ml)

IMMUNITY IN COPPER DEFICIENCY

275

of the cell suspensions from spleen were layered on top of 3ml Ficoll-paqle (Pharmacia Fine Chemicals, Piscataway, NJ) and tubes were centrifuged at 400 x g for 30 minutes at 4~ Lym~hocytes were carefully removed from the interface. Splenic lyn~hoid cells, cervical l y ~ node cells (CINC) and thymocytes were each centrifuged (400 x g for i0 m/n) and washed 3 times with cold RR41 1640. After resuspension of the final cell pellet, total ~id viable cell numbers were determined. Tne suspension was adjusted to 4 x i0 cells/ml=and 50ul cell suspension, 100ul RPMI 1640 containing 2-m~%0toethanol (5 x 10-~M) and 4% heat-inactivated rat serum, and 50ul medium containing the indicated amount of mitogen were added to each well. Replicate sets of cells from each organ of the donor rat were incubated in m ~ i u m containing pooled sera that had been prepared frum groups of rats fed diet containing the same carbohydrate and either adequate or deficient copper. $~en the copper status of the lymphocyte donors and sera were identical, the test system was defined as humologous. ~hen cells and sera were obtained f~um animals fed diets with diffe/ent levels of dietary copper, the test system was defined as heterologc~s. Lymphoid cells were incubated with concanavalin A (Con A) and phytohemagglutinin (PHA) in final concentrations ranging from 0.2 to 3.12 ug mitogen per well. Only data from control cultures (no mitogen) and optimal levels of each mitogen are reported. All tests were performed in triplicate. After 48 h~urs incubation in 95% air-5% OD 2 at 37~ 0.5 uCi 3H-thymidine (~H-thy; 6.7 Ci/nm~le, New England Nuclear Products, Boston, MA) in 20 ul ccmplete medium was added to eadl well. Cultures were incubated for an additional 24 hours before collection of acid precipitated material on glass-fiber fi~ters (Cell Harvester Model 7019, Skatron Inc. Sterling, CA) and the amount of ~H retained on the washed and dried filters was determined. Sti~alation index (SI) was calculated as mean ~ ~H per culture at optimal concentrations of mitogen divided by mean qsm ~H per culture without mitogen. Analyses. The activity of O/,Zn-superoxide disrs/tase in isolated cells from lymphoid tissue was determined spectrophotumetrically by measuring the inhibition of the autoxidation of pyrcgallol as described by ~ et al. (6). ~ z y m e activity was completely inhibited by 1 mM Nard, thus confirming efficient ekTx~ction of Mn-superoxide ~ . Total cell protein was determined ~y a modified Lowry assay (7) using bovine serum a l ~ m d n as standard. After sonication, aliquots of isolated thymocytes were digested by a combined dry and wet ashing method (8). Copper was determined by atomic absorption spectrcmetry ( P e r k / n - ~ Model 5000, Norwalk, CT) using an air-acetylene flame. Identical handling and analysis of certified Standard Ref~ Material rice flour 1568 (National Bureau of ~ r d s , Gaithersburg, MD) demonstrated that the recovery of copper exceeded 95%. Data analysis. Heterogeneity of varianoe was corrected by log transformation of values for incorporated ~H-thy. Data were evaluated by analysis of variance. Student-Newman-Keuls (SNK) multiple range tests were used to determine significant d i f f ~ (p<0.05) between treatment means (9). RESULTS Althcu~ serum copper was depressed similarly in rats fed F-Cu and S-Cu diets, only those fed the F-Cu diet had enlarged hearts and lower hematccrits than their copper-supplemented controls (Table i).

276

U. BABU and M. FAILLA TABLE 1

~hole Body and Heart Weights, Hematocrit and S e m ~ Oq~per of Rats Pair-Fed Diets Containing Either Fructose or Starch with Adequate or Deficient Copper Parameters

Fructose +Cu

Body weight (g) Heart weight

202 + 6 a

Starch --Cu

214 + 4 a

4-(3/

219 + 5 a

-O.1

221 + 4 a

0.36 + 0.01 b

0.57 _+ 0.02 a

0.36 + 0.01 b

0.40 + 0.01 b

42.0 + 0.8 a

32.6 + i . ~

40.3 _+ 0.5 a

39.0 + 0.3 a

108 + 4 a

i0 + 1c

99 + 3b

9 + ic

(g/10~) Hematocrit (%) Serum copper (ug/1OOml)

Values are means + S.E.M. (N=6-7 per group). Significantly different values within a row are indicated by presence of different letters as

~ip~. Splenic and cervical ly,p h node masses were independent of dietary copper and carbohydrate (Table 2). The thymus was significantly smaller in rats fed the F-Cu diet than in the other dietary groups. The impact of dietary copper deficiency on the copper status of lymphocytes, as assessed by the activity of Cu, Zn-superoxide dismutase (SOD), was more sever~ in rats fed diets containing fructose compared to starch. SOD activities in splenocytes, CINC and thymocytes from rats fed F-Cu were 58, 60 and 30% lower, respectively, than those in the respective cells from rats fed F+Cu diet. Enzyme activities were 31, 45 and 15% lower in s p l e r ~ , CINC and thymocytes, respectively, from rats fed the S-Cu diet than in cells frcm rats fed S+Cu diet. similarly, the concentrations of copper in thymocytes from rats fed F-O/ and S-Cu diets were 61 and 32% lower, respectively, than tho6e in cells faum their copper-supplemented controls. Copper levels were not measured in splenocytes or CINC because of the lack of sufficient material. The yield of thymocytes was slightly lower in rats fed -Cu diets than +Cu diets; yields of s p l ~ and CINC were not affected by dietary treatment. Splenocytes from F-Cu rats incorporated only 40 and 29% as r~/ch ~ - t h y into DNA upon exposure of cultures containing homologous sera to optimal concentrations of Con A and I~A, respectively, as oells from rats fed F+Cu diet (Table 3). However, mitogen-irn3/ced DNA synthesis in splenocytes from rats fed S+Cu and S-Cu diets were not significantly different. This influence of dietary carbohydrate on the mitogenic responsiveness of splenocytes frum rats fed -Cu diets was also observed at suboptimal ooncentz-ations of Con A and PHA (data not shown ) . DNA synthesis in splenocytes from rats fed +Cu diets was not affected by dietary carbohydrate. JH-thy incorporation was 15-50% higher in unstimulated cultures of splenocytes from rats fed -Cu diets than in those from rats fed +Cu diets (Table 3). Consequently, stimulation indices of splenocytes fill, S-Cu rats, as well as frcm F-Cu rats, were significantly lower than those of cells fr~n +Cu controls.

IMMUNITY IN COPPER DEFICIENCY

277

TABLE 2

Effects of Diets on Mass of L y ~ i d O r g a n s Cu,Zn-SupercxideDismataseActivityof

Parameters

Fructose +Cu

Spleen mg/100 g b o d y w t s p l ~ xl0-b/g SOD (U/mg protein) Cervical lymph nodes mg/100 g ~ody wt CINC xl0-~ SOD (U/mg protein) Thym~ mg/100 g body _w~ thymocytes xl0-~ SOD (U/rag protein) ug Cu/g cell protein

and the Yield and Isolated Cells

199 • 7a 207 • 15a 10.6 • 1.9 a

112 + 13 a 208 + 41 a 6.2 ~ 0.8 ab

291 1537 5.4 6.6

+ + + ~

Sta~c/~ -Cu

20 a 148 ab 0.4 b 0.5 ab

+Cu

-Cu

195 • 14a 182 • 22 a 4.5 • 0.5 c

205 • 15a 168 + 21 a 8.9 ~ 0.8 ab

180 • 6a 185 + 27 a 6.1 ~ 0.6bc

121 + I0a 252 • 39 a 2.5 + 0.5 c

120 + 12 a 246 _+ 38 a 8.9 + 1.2 a

I01 + 15a 249 + 28 a 4.9 -_+ i.ibc

212 1220 3.8 2.6

+ + ~ +

21b i17 ab 0.5 c 0.6 c

321 1714 7.2 8.4

+ + + +

Iia 173 a 0.6 a 1.0 a

282 1049 6.1 5.7

+ + + ~

iia 122 b 0.4 ab 0.7 b

Values are means + S.E.M. Cell numbers represent yield isolated from lymphoid organs by standard procedures as outlined in Materials and Methods. Different letters as superscripts within a row indicates that the groups differ significantly at p<0.05. In medium with homologous sera, both unstimulated an~ Con A-stimulated CINC from rats fed -Cu diets incorporated significantly more JH-thy into DNA than cells from rats fed +Cu diets regardless of dietary carbohydrate (Table 4). Mitogen induced DNA synthesis was greater in cells from rats fed F+Cu diet than in those from rats fed S+Cu diet; DNA synthesis in CINC frc~ rats fed -Cu diets was not influenced by dietary carbohydrate. Tne differential reactivity of CINC from rats fed -Cu and ~ diets was independent of the concentration of Con A in the incubation medium (data not shown). Con A-stimulated DNA synthesis in thymocytes isolated from rats fed F+Cu and F-Cu diets was similar in medium containing homologous sera (Table 4). However, the stimulation index at the optimal dose of the mitogen was significantly higher in cells f r ~ the F+Cu group than for cells from the F-Cu group because of differences in ~H-thy incorporation in unstimulated cultures. Incorporation of 3H-thy by unstimulated and Con A-stimulated cultures of thymocytes from rats fed starch diets was not affected by dietary copper. DNA synthesis in unstimulated cultures was lower when cells fz~Y~ rats fed -Cu diet were incubated in medium with 2% sera fL~u rats fed +Cu diet compared to sera from rats fed -Cu diet (Fig. i). Conversely, s p l e ~ and thymocytes from rats fed F+Cu diet incorporated significantly higher levels of

278

U. BABUand M. FAILLA TABLE 3

Effect of Dietary TreatEerfc on Reactivity of Splenocytes to Con A and I~A

Mitten

Diet

3H-thy incorporation

SI

Mi~en

emt~1 c p m x 10-2 ConA

F+Cu F-Cu

s~ s~ PHA

F+Cu F-Cu S+Cu S-CU

5.2 • 0.i ab 7.9 + 0.9a 4.8 u o.6b

1233 • 42a 494 + 9 ~ a b 894 ~ 15s

236 • 5~ 61 + 183 ~ 11b

s.s~o.v ~

v86~143 ~

138~12 c

4.3 6.0 4.4 6.8

452 129 477 309

108 20 108 44

• • • •

0.4 a 1.3 a 0.4 a 0.8 a

• + ~ ~

36a 41b 62a 61a

• • • •

5a 2c ~0a

4D

Splenocytes were ircubated ~ medium containing 2% homologous sera. Values (means + S.E.M. ) for ~H-thy inoorporation are those for cells incubated in separate plates containing medium with either no mitogen or the optimal level of either Con A (0.39) or I~A (l.56t~g) per well. SI, i.e., sti~/lation index, was calculated as described in Materials and Methods. The presence of a different letter as superscript within the same column indicates that means differ significantly for the indicated mitogen at p<0.05. 3H-thy in mitogen-free cultures containing sera from F-Cu rats as compared to homologous sera. The source of sera did not significantly affect E~A synthesis in unstimulated cultures of cells from rats fed S+Cu diet. similarly, the type of sera did not affect mitogen-induced ~ synthesis in cultures of ~ l ~ x ~ / ~ s , c ~ c ~ d t h y m ~ y ~ s (~ta not ~x~n). DISCUSSION The results of this study show that the in vitro responsiveness of T-cells from various lymphoid organs to mitogens is differentially affected by dietary copper deficiency in young male rats. Con A-induced DNA synthesis was lower in spler~, higher in CLNC and unaffected in thymocytes isolated from rats fed copper-deficient diets than that in the respective cells from rats fed ~lemented diets. In contrast with this tissue-dependent responsiveness to the T-cell specific mitogens, DNA synthesis in unsti~ulated cultures was usually higher in cells isolated from the organs of rats fed copper-deficient diets than that in copper-supplemented controls. These observations are in general agreement with others. Several groups have reported decreased mitogenic reactivity of splenocytes from copper-deficient rodents (3,10,11). Davis et al. (ii) recently reported t~at cultures of CINC f~i, cc~per-deficient rats incorporated higher levels of ~H-thy than those from control rats in medium containing optimal levels of Con A. Lukasewycz and

IMMUNITY IN COPPERDEFICIENCY

279

TABLE 4

Effect of Diets on Reactivity of CLNC and Tnymocytes to Con A

L!a~mhocytes

Diet

3H-thy m r [ o r a t i o n Control

SI

+Con A

cpm x 10-2 CINC

F+Cu F-O/

3.1 + 0.Ib 8.3 + 1 1 ~

2.9 7.6 Tnymocytes

F+Cu F-Cu S+Cu S-Cu

1.5 3.0 3.0 3.3

1.2 a + + + ~

0.2 b 0.4 a 0.7 a 0.5 a

813 + I02b 1 2 2 8 + 164a

263 + 24 a 148~ ~b

s32 ;- 43~

186 7-

1440 ;- 191;'

195 -T-

35 42 66 59

+ + + ~

7b 3ab 16a 6a

23 15 22 19

+ ia + 2b _+ 2 a + 2 ab

Isolated cells were ~ t e d in medium containing 2% pooled sera obtained from rats fed the same diet as lymphoid tissue donor. Values for ~H-thy incorporation in the presence of mitogen for cttltures containing 0.39 and 0.2ug Con A with CINC and thymocytes, respectively. Different letters as superscripts within the same column indicates that cells from different dietary ~--roupsdiffer significantly (p<0.05) for that type of lymphoid cells. (12) have reported that dietary copper status did not influence DNA synthesis in unstimulated and mitogen-stimulated cultures of murine thymocytes. One of the novel findings of this study was that the type of dietary carbohydrate modulated the mitogenic reactivity of splenccytes from copper-deficient rats. Splenocytes frum rats ~ed the oopper-deficient diet with fructose incorporated significantly less ~H-thy when treated with Con A and PHA than oells from rats fed the copper-supplemented diet with fructose. In contrast, dietary copper status had a minimal affect on mitogen-irduced DNA synthesis in splenocytes frcm rats fed starc~h diets. We have also found that the humoral irmm/r~ response of rats injected with sheep erythroc~tes is impaired to a greater extent when rats are fed a copper- deficient diet oontaining fructose instead of starch (4). In addition to these carbohydrate dependent effects of dietary copper deficiency on the ~ function, cardiac hype~y and reduced hematocrit were only observed in rats fed[ F-Cu diet (Table 1 and ref. 4). Dietary copper deficiency in young rodents is associated with reduced concentrations of this micronutrient in serum, thymus and spleen (Table 1 and refs. 4,13) and in isolated t h y m ~ (Table 2). similarly, the activities of the c u p r o e n ~ cytodL---nme oxidase and superoxide dism.fcase are decreased i n spleen and thymus (6,13), thymocytes (Table 2 ar~ ref. 12), spler~"ytes (Table

280

U. BABU and M. FAILLA

SPLENOCYTES r 0

900

,m

CLN CELLS 900

THYMOCYTES 400

800

K.

0

8O0

#

700

L_

0 0 C

700

~

0 C

600

9 --

E

300

sool sooi 400

200

500

r

300

I

4OO

19

200

E

11. U

300

100

Homologous Heterologous

100

Homologous Heterologous

Homologous Heterologous

Type of test system

FIG. 1 Influence of serum source on DNA synthesis in unstimulated cultures of splenocytes, CINC and thymocytes from rats fed F+Cu (O), F-Cu (O), S+Cu (O) and S-Cu (m) diets. Cultures centained medium with 2% pooled sera that had been obtained from rats fed diet with the same (homologous system) or a different level of copper (heterologous system) than the lymphoid cell donor. ~ presence of an asterisk above the3symbol in the homologous system cel~m~ of each panel indicates that H-thy incorporation in the two test systems was significantly different at p<0.05. 2 and ref. ii) and CLNC (Table 2). However, the decreased copper status of cells frcm thyrm/s and cervical lymph nodes was not correlated with suppressed mitogenic reactivity (Tables 2 and 4, refs. 11,12). The cultures used in this study to assess T-cell responsiveness to mitogens contained oell types other than lymphocytes, including sume cells that affect lymphocyte function. ~hus, the different mitoge/~c responses of T-cells from the three ly~shoid tissues may be due to organ specific effects of chronic dietary copper deficiency on the types, relative numbers, degree of differentiation and function of l y m ~ themselves, as well as the activities of various effector cells. Lukasewycz et al. (3) have reported a higher ratio of B to T-cells, a decreased population of T-helper cells, and diminished maturation of B oells in spleens of mice fed copper-deficient diets. Moreover, the lipid and protein ~ i t i o n of p l a ~ membranes from lymphocyte-enriQhed preparations of splenocytes,

IMMUNITY IN COPPERDEFICIENCY

but not thymocytes, was altered by copper deficiency (14). This alteration in plasma membraI~ ~ i t i o n was correlated with impaired ability of splenocytes from copper-deficient mice to stimulate allogeneic control cells in a mixed lyrmphocyte reaction (15). Also, ilxm/bation of s p l ~ from control rats in a copper-deficient medium decreased the production of T-cell replacing factor in a mixed lymphocyte reaction (16). In addition to these apparently direct effects of ~ deficie/%c~y on ly~ocytes, Davis et al. (ii) have suggested that the reduced mitcgenic responsiveness of splenocytes fz~L rats fed a copper-deficient diet is actually due to an impairment of an enhancer activity of macrophages. The decTeased production of interleukin-i by splenocytes incubated in cc~per-deficient medium (17) and lower thymic factor activity in serum of copper-deficient rats (2) support the possibility that inadequate copper nutriture adversely influences effector cell activity. More information on the effects of copper deficiency on both the l y ~ o c y t e s and their effector cells within each lymphoid organ is required to understand the tissue specific responses of lyr~ocytes to mitogenic activation. Increased incorporation of 3H-thy in unstimulated cultures of splenocytes frc~ copper-deficient rodents has been previously reported (3,10,11). We found ~ t oopper status of serum donors tended to modulate basal incorporation of H-thy in ly~phocytes from fructose fed rats and also in rats fed S-Cu diet. In general, sera from .c~.-~.ficient and control rats increased and decreased, respectively, H-thy incorporation in unstimulated cultures. Tnis observation suggests that dietary copper status influences the level(s) of one or more soluble factors that affect DNA synthesis in lymphoid tissue. Further research is needed to identify the(se) factor(s) and the responding cell type(s). Because DNA synthesis in cultures containing optimal concentrations of mitogens was not significantly altered by the origin of sera, chronic copper deficiency also appears to influence the mitogenic responsiveness of T-cells from spleen and oervical lymph nodes in some manner that is independent of serum factors.

Drs. Tim Kramer and Tom Caperna provided valuable instructions on studying mitogenic reactivity during the initial stages of the study. Th~ assistance of Richard Barr in animal care and the secretarial assistance of Christina Woods and Shirley Weese is most appreciated. REFERenCES i. ~ k a JR, I//kasewycz OA. Cc~3er deficiency suppresses the irmm/r~ response of mice. Science 1981; 213:559-61. 2. Vyas D, Chandra RK. Tnymic factor activity, lymphocyte stimulation response and antibody producing cells in copper deficiency. Nutr. Res. 1983; 3:343-9. 3. Inkasewycz OA, Prohaska JR, Meyer SG, Schmidtke JR, Hatfield SM, Marder P. Alterations in lymphocyte subpopulations in copper-deficient mice. Infect. ~ . 1985; 48: 644-7.

281

282

U. BABUand M. FAILLA

4. FaillaML, BabuU, Seidel KE. Use of imm/noresponsiveness to demcnstrate thatthedietaryrequirement for co,per in ycungrats is greater withdietary f r u c t o s e ~ d i e t a r y s t a r c h . J. Nutr. 1988; 118:487-96. 5. American Institute of Nutrition. Report of the AIN Ad H~c Cumnittee on ~ for nutritional studies. J. Nutr. 1977; 107: 1340-8.

6. Pr~mska JR, D~ning SW, Lukase~cz OA.

~u~nic dietary

deficiency alters biochemical and morphological pruperties of mouse lymphoid tissues. J. Nutr. 1983; 113: 1583-90. 7. Nerurkar iS, Marino PA, Adams DO. Quantitation of selected intracellular and secreted hydrolases of macrophages. In: Herscowitz HB, Holden }[9, Bellanti JA, Ghaffer A, eds. Manual of macrcphage methodology. Marcel Dekker, Inc. New York: 1981; pp. 229-58. 8. Hill AD, Patterson KY, Veillon C, Morris ER. Digestion of biological materials for mineral analyses using a combination of wet and dry ashir~. Anal. Che~n. 1986; 58:2340-2. 9. PetersenRG. Design andanalysis of experiments. Inc. New York, 1985: pp. 429.

Marcel Dekker,

i0. Lukasewycz OA, ProhaskaJR. Ly~0hocytes frcm ccpper-deficientmice exhibitdecreasedmitogen reactivity. Nutr. Res. 1983; 3:335-41. 11. Davis MA, Johnson WT, Briske-Anderson M, KramerTR. LyI~phoidcell functions duringcopperdeficiency. Nutr. Res. 1987; 7:211-22. 12. I//kasewycz OA, ProhaskaJR. Effect of postweaning copper deficiency on thymocyte activity in mice. Fed. Proc. 1987; 46:590. 13. ProhaskaJR. Changes in t i s s u e ~ , concentrations of copper, iron, c ~ oxidase and superoxide dismutase subsequent to dietary or genetic copperdeficiency in mice. J. Nutr. 1983; 113:2148-58. 14. KorteJJ, Prohaska JR. Dietary copper deficiency alters protein and lipid cc~position of rmlrine lymphocyte plasmamembranes. J. Nutr. 1987; 117:1076-84. 15. Lukasewycz OA, Kolquist KL, Prohaska JR. Splenocytes frum copper-deficient mice are low responders and weak stimulators in mixed l y ~ o c y t e reactions. Nutr. Res. 1987; 7:43-52. 16. FlynnA, Yen HR. Mineral deficiency effects on thegeneration of cytotoxic T-cells and T-helper cell factors in vitro. J. Nutr. 1981; 111:907-13. 17. Flynn A, ioftus MA, Finke JH. Production of interleukin-i and interleukin-2 in allogeneic mixed l y ~ o c y t e cultures under copper, magr~=sium and zinc deficient conditions. Nutr. Res. 1984; 4: 673-9.

Accepted for publication November 8, 1988.