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Effect of total calorific deprivation on host defence in the horse
Pesearch in k
brrect or total calorific deprivation on host defence in the horse J. KENY Department of Clinical Studies, vererrnary acnoor, unrversrry oj rennsyrvanra, jar w. arreer Koaa, Kennett Square, Pennsylvania, 19348, USA (orses we]re deprived of foodI but not water for ~eriodof f ive days. 7rhere was a progressive decrea: n circulatiing lymph ocyte cou nt and cc~mprornisc -. . or yeas1 . ~[err. iro rn lost defence. x-..z.-. In vrrrv pnagocyrosls I mean of 97 per cent in fed horses to 78 per cent i~ n lorses deprived of food for five days. The ArthlJS esponse tc intradermal challenge with allergens ft''I o 57 per cent of fed values. T'hese changes indica hat food deprivation increase:s the susc~ eptibility 1 lacterial infections; persistent anorexia in chron lacterial infections may limit the horse's ability 1 ast off inf~ection. -LA-.
.-?.
- @ -
-
@
..@
-
of refeed ing a ha1f maintenlance ratilon was ed; therea~fter,a m.aintenanct: ration Hlas fed. lies were c:ollected 16 hours pc st prandi ally 24, ". ,--..-. 1L-. ..-c--A:- ---:...I ,--,?-A 45, I L nours lnro [nc: r c : ~ t : t : u ~ r ~ r UCIIUU ( I r l r u samples). One horse became ill or;thk first day of refeelding and v/as remove:d from t h:~experime,nt. I---
Skln testing was perrormea us ique and allergens described by Halliwt ). Each skin test involved the injection of ;tarnine-positive control, saline-negative control Lllt: anu 214 allergens. Skin testing was performed 0 1 1 *Lfirst f'ed da; th e last day of fasting and the si.xth 'day ie of re:feeding. Six experimental hlorses we1re skin I N people and labc ~imalsprc - L - " ..A. -- L testecI. An equa 1 number of fed cont.rol horses kept at ~ ~ ~ I I I IUI I L U III I I ~ S~ U V C ~ S ~ ITCLS : UII I I V S L U C I C I I Ce. ~ re were also skin tested with tne same Ilnterva Id pastu rnpaired i mmunoglcobulin (Law et al 1974) an betwf :en tests. The diameter of the reactions t o these ornplemer~tproducti on (Sirisin ha et al 1973, Dioni. allergens was measured at 30 and 240 minutes; with a t al 1977)., cell imrnunity (Smythe et . me(iiated . - - .. ruler. When possible measurement was perfor med by 971, McFarlane and H a m ~ d 1973, Dionigi et ... traine:d personnel ignorant of the experlimental 977), neutrophil chemotaxis (Dionigi et a1 1977) an treatrnent and the purpose of the experimer~ t The . eutrophil bactericidal activity (Seth and Chandr.a mean response per allergen was calculated fc )r each 972, Palmblad 1976) have been reported. Most c, horse for the 30 and 240 minute time periods . These hese studies have involved long term partiia1 were used as an index of type I o r imr .. . nea~ate eprivation s of either protein o r energy . In kquinle sensitivity, and t ype 111 or. Arthus hyper0 hyper that eat n ractice it :is not unusual to see animals sensit ivity reactiions respec:tively. horst:s 30d. for a ..r elatively short period. For exalmple .. " . ~t each sampllng blood was collected Into EDTA re held off feed before surgery; severe infections can Iso induce anorexia. This report dealIs with thle and 1ithium heparin tubes. Differential wh ite cell ffects of short term (five day) total derxivation c)f count s were performed on EDTA treated bloo d using a ma~dified Wright's stain method (Diff Quick; aod on host defence in the horse. Harle co). White blood cell counts were quar~titated with an automated counter (Coulter Mod el ZF; laterials a1nd methodIs Coultc:r Electronics). Neutrophil phagocytosis was subjec:ted to toti The.. man, o f.seven . agement . . . ho rses . assaw:d in vitro using heparin treated blood cc~llected ....e alorltlc deprivation has been d e s c r ~ ~ eelsewher d four of the horses. Blood was centrifulged at : from Vaylor et a1 1980) and is summarised here. Horsr for 15 minutes. Plasma and buffy coa~t were 'ere acclimatised to a maintenance diet of a 5 0 0 g ted separately into plastic tubes and thc: buffy collec Dmplete pelleted feed. At the start of th rrr\"t ias resuspended in 0 . 5 ml homologous p.lo..-.-. ! * ' . > , ' . a . Xperimental period, blood samples were collected 1 is suspension 0 . 5 ml o f a lOOx 106/ml ours post prandially at 08.00 for three consecutiv ~ s i o nof boiled baker's yeast in phc~sphate aYs (fed samples). Feed was withheld for five da) red saline was added. The suspensioIn was nd s a m ~ l e were s collected at 40.64, 88, 112 and 13 r n l x e u on a vortex and incubated at 37OC. Aft1tr V, n L, I n the fir! ours post r (fasted s jt 5, 10 and 20 minutes the mixture was vortex,ed and samples were taken with a Pasteur pipette. !Smears a s : Departmcnt or Vcfterinary Clinical StudieS, an, SN7 0WC) Saskatchewan, Saskatoon Saska~chcw were..- made o n microscope slides, stainec1 with --..-"" ied Wright's strain and tht: percent;3ge of modlf Pracnt addrC ~ S .c ~ ar~rish u Embassy, PO Box 801. Khanoun1, hdan. neutrc)phils cont aining yea.st countedI at each ti)me. ....I-..*-:A:-
rn,radermal
. . A -
:f ,f
.
..
! - .
--
.
.
.
Statistical analysis 1was perform e d usinl;a pockett lculator . - . w. i t h statisti - ics modulc:(TI. 59 F'rogramm.11ecalculator; I exas Instruments). I nere was n o ~nificant difference between fed day results for :her haemograms o r neutrlophil phagocytosis ,tectable with theI two-tailled paired t test. c-- L I I eref fore, mean valucb, IUI ~ ~ a e m o g r a m variables and neutrophil phagocytosis, were calculatLed for fecI da.ys and were used in subsequent compaIkens wittI fa:sted days using the two-tailed paired t test. At the first skin test the control and experimental groups of horses had similar sk;in test r eictions foir both the 30 and 240 minutes respons,es ( t testt P>O.l). For each group of norses significani anges in the skin test respionse to s;ubsequenl allenges u'as compared using two way 1Inova anc uncan's ne:w multipl e range test (Steele ;and Torrie 160).
-
.a"
P0.01. Thic" change was also significant when fasted values for t he~ experimental grol~p were clompared to the
..
7-
-r
..
. ....
-8
I
T
T
T
suits
Total mat ure and ~ I I I I I I ~ L U I~leutrophil C counts dc jt change significantly with fasting (Fig 1). D u r i n ~ ie days of total calorific deprivation blooc rnphocyte counts fell by one third and ther covered somewhat during the refeeding perioc 72 ig 2). 1 Neutroph il phagoc! rtosis decllines durir~g fasting sfeeding Hours c,f food depri!lation Fig 3 the time cours;e of the plhagocytic Iresponse i!j .-..-. , .L--. __,mparearor iea, rive aay iooa aeprlveu anu rnrrc rlcl L: local Diooa lvmDnocvte counrs. meanzstanaara aevl iifferent day refed horses. Day to day changes in the 2() five horries. Values m:arked with arI asterisk are! :0.05 using 1'he paired I te minute phagocytic index are shown in Fig 4. During: from fecI values at P< fasting neutrophil phagocytosis fell significantl) f-3m a mean of 97 per cent in fed horses to 78 pel nt, after five days of food depr IThe skin test res]ponse t o histamine:, did no ttests withii ~ d e r g osignificant ch anges bet! ween. skin I . ther the experimental or control horses (Anov:
.
- 3 fl
3
>.--?..->
--J
11
I
-
[ ' I F I
A X Horses Five dayfed fasted
ree day refec
I
-
1
4
1
1
1
1
88 112 136 24 48
1
72
-\
w n of food
;of
deprivation
refeedin
IG 1: Total blood neutrophil (mature a1nd immature]I counts in fi! ~rses,data are expressed as meanestan dard dwiation . None of tt "t..... ..-:~luesare significantly different from f hA warus> urlllll palrca ... r,
..
'"0
XxX
d
V
V
Y
5
10
20
Time (minutes) C l U 5 : I I Ime course of the in vitro phagocytic response in four horses. Data arc:expressed a! ~darddeviation
371
Host defence in food deprived horses
second skin test in the control group using the f test, p = 0.01 i
ussion
..
. .. . .
.
Peripheral blooa lyrnpnocyfe counts re11 aurtng total caloric deprivation. Similar observations have been made in dogs (Dionigi et al 1977) and malnourished sick people. Rats fed protein dleficient cells in diets have decreased numbers of lymphoid ,.--. spleen and thymus (McEarlane andI Hamid 15113). ~n this study blood neutro]phi1 concc:ntration 1l ~ a snot affected by fasting. Th is is simiilar to re sults in *--,\ . ~. . healthy people (Palmblaa IYIO), m conrrasr caloric :reases blo deprivation dec od neutrophil count in dogs (Dionigi ~ et al 1977) ~ Decreased neutrophil s not seer phagocytosis i: I in healthy fasted people n7-n VL pL.dple with protein calo-;a-n1'L1b L l l a l .he2 eta1 17 1 tion (Seth and Chandra 1972) but did (:xcur in study along with a decrease in the Arthus mse. Decreased concentrations of comldement . .et. a1 proteins are seen in calorie deprived dogs (Dion~g~ 1977). There are decreases in a variety of comvlement proteins in protein-calorie malnourished people (Smythe et al 1971, Sirisinha et al 1973). Similar reductions in complement in horses could explain changes in neutrophil function and Arthus reactivity observed in this study. Complement is involve!din the Arthus reaction and may assist in the opsonis:ation of yeast by neutrolphils. We have doc:umented marked dc:creases in several arms of the ho!;t defence system following shc)rt term -- -,-I-:1L a :--I food de~rivatiurr. I 111srlab I I I I ~ U I L ~ I Il~~t~lications L horse W I:Ifare. P eriods of electivc: food ivation d uring tra nsport or for vet erinary oses shor~ l dbe kc:pt as short as p~ossible. .exia commonly accompanies illness in r i r b horse:s and ef forts to iimprove 1'ood intake may bolster host derfence.
.
.
. . 1
-
.
1
2
noun of refeedinc
"-.-T
leprivation
~-
IFIG 4: Neut rophil phagc Imean* standl , . . . ~n . ,lour . norses. values marked!O.wminutes. .t h. an asterisk oevtarlon are sign... nt from fed v,slues at P(O.05 using the paired t test
.
Test horses
- mtrol horses
1
A . *
... ,.
II.1".
This work was supported in part by the Philsdelphia Foundation and Biomedical Rpcparrh No SO7 RR 5464-15. - ort Grant USPHS - - -
Rec,eivedforp
u .
allergen -L-Il---
8 , -
IC
FIG 5: Conlparison of the Arthur response iln control and experimental horses. Cc~ntrol horsc:s were fod through01ut. Experimental nones wcre fed at first a llergen - . tor - challi .:nge, fasled. * ,1br 'our davs at the second challenge and reled ltve days at rne rnlro ere were st% horrcs in both experimental and control
-
...
January 2
References
Third
First allergen
I
DIONIGI, R., ZONIA. A., UOMINIUNI, L., CiNtS. b. (e RAl-LABIO, A . (1977) Annals of Surgery 185.467-474 -. -. HALL.IWELL, R.E.W., FLEISCHMAN, J.B., MACKAI'-SMITH, M., BEECH. J. & GUNSON. D. (1979) Journal of the American Vertprinary Medicine Association 174. 277-281 LAW. D.K., DUDRICK, S.J. & A B W U , N.I. (1974).Annals of c. Jurrerv 179. 16%-173 McFA & HAMID, Imn 153-161
American Jo
ELD, D.S. & ACLAN! srinary Resea,rch 41,899-9
\LMBLAD,
rndinavian
~ Y L O R , J.I
-.- --.
Jc)urnal oJ Hat
1)
'
LI I-LLO
~ A N C H E Z , A,, K L L ~ E K , J . L . , L A U , ti.>., I A ~ I A U , r.1 ., WILLARD, R.E., McMILLAN, P.J., CHO, S.Y., MAGIE, A.R L. ®ISTER, U.D. (1973) American JournaloJClinicalNulrilion 26, 1180-1184 CHANDRA, R.K. (1972) Archives of Dkemes in
ID, R., E D E L M A N , R . , S I R I S 1 N H A , S. ILSON. R.Ei. (1973) Lance1 i, CHA RUPATANP 1016- 1020 JLVl l 1 rIE, P.M., SCHONLAND, M., BERETON-STILE?3. U.U.. ' COO VADI, H.h4 . . GRACE1, H.J.. L(3ENING. \N.E.K., MAFIOYANE, A,,, PARENT. IM.A. & VOS , G.H. (1971) Lancet ii, 935)-944 ocedures STEEL1E, R.G.D. &TORRIE. J.H. (1960)Prir1,.,.,,~.... o f Statistics. New York, McGraw-Hill. p 107 0. ' " T T
...
brrect or total calorific deprivation on host defence in the horse J. KENY Department of Clinical Studies, vererrnary acnoor, unrversrry oj rennsyrvanra, jar w. arreer Koaa, Kennett Square, Pennsylvania, 19348, USA (orses we]re deprived of foodI but not water for ~eriodof f ive days. 7rhere was a progressive decrea: n circulatiing lymph ocyte cou nt and cc~mprornisc -. . or yeas1 . ~[err. iro rn lost defence. x-..z.-. In vrrrv pnagocyrosls I mean of 97 per cent in fed horses to 78 per cent i~ n lorses deprived of food for five days. The ArthlJS esponse tc intradermal challenge with allergens ft''I o 57 per cent of fed values. T'hese changes indica hat food deprivation increase:s the susc~ eptibility 1 lacterial infections; persistent anorexia in chron lacterial infections may limit the horse's ability 1 ast off inf~ection. -LA-.
.-?.
- @ -
-
@
..@
-
of refeed ing a ha1f maintenlance ratilon was ed; therea~fter,a m.aintenanct: ration Hlas fed. lies were c:ollected 16 hours pc st prandi ally 24, ". ,--..-. 1L-. ..-c--A:- ---:...I ,--,?-A 45, I L nours lnro [nc: r c : ~ t : t : u ~ r ~ r UCIIUU ( I r l r u samples). One horse became ill or;thk first day of refeelding and v/as remove:d from t h:~experime,nt. I---
Skln testing was perrormea us ique and allergens described by Halliwt ). Each skin test involved the injection of ;tarnine-positive control, saline-negative control Lllt: anu 214 allergens. Skin testing was performed 0 1 1 *Lfirst f'ed da; th e last day of fasting and the si.xth 'day ie of re:feeding. Six experimental hlorses we1re skin I N people and labc ~imalsprc - L - " ..A. -- L testecI. An equa 1 number of fed cont.rol horses kept at ~ ~ ~ I I I IUI I L U III I I ~ S~ U V C ~ S ~ ITCLS : UII I I V S L U C I C I I Ce. ~ re were also skin tested with tne same Ilnterva Id pastu rnpaired i mmunoglcobulin (Law et al 1974) an betwf :en tests. The diameter of the reactions t o these ornplemer~tproducti on (Sirisin ha et al 1973, Dioni. allergens was measured at 30 and 240 minutes; with a t al 1977)., cell imrnunity (Smythe et . me(iiated . - - .. ruler. When possible measurement was perfor med by 971, McFarlane and H a m ~ d 1973, Dionigi et ... traine:d personnel ignorant of the experlimental 977), neutrophil chemotaxis (Dionigi et a1 1977) an treatrnent and the purpose of the experimer~ t The . eutrophil bactericidal activity (Seth and Chandr.a mean response per allergen was calculated fc )r each 972, Palmblad 1976) have been reported. Most c, horse for the 30 and 240 minute time periods . These hese studies have involved long term partiia1 were used as an index of type I o r imr .. . nea~ate eprivation s of either protein o r energy . In kquinle sensitivity, and t ype 111 or. Arthus hyper0 hyper that eat n ractice it :is not unusual to see animals sensit ivity reactiions respec:tively. horst:s 30d. for a ..r elatively short period. For exalmple .. " . ~t each sampllng blood was collected Into EDTA re held off feed before surgery; severe infections can Iso induce anorexia. This report dealIs with thle and 1ithium heparin tubes. Differential wh ite cell ffects of short term (five day) total derxivation c)f count s were performed on EDTA treated bloo d using a ma~dified Wright's stain method (Diff Quick; aod on host defence in the horse. Harle co). White blood cell counts were quar~titated with an automated counter (Coulter Mod el ZF; laterials a1nd methodIs Coultc:r Electronics). Neutrophil phagocytosis was subjec:ted to toti The.. man, o f.seven . agement . . . ho rses . assaw:d in vitro using heparin treated blood cc~llected ....e alorltlc deprivation has been d e s c r ~ ~ eelsewher d four of the horses. Blood was centrifulged at : from Vaylor et a1 1980) and is summarised here. Horsr for 15 minutes. Plasma and buffy coa~t were 'ere acclimatised to a maintenance diet of a 5 0 0 g ted separately into plastic tubes and thc: buffy collec Dmplete pelleted feed. At the start of th rrr\"t ias resuspended in 0 . 5 ml homologous p.lo..-.-. ! * ' . > , ' . a . Xperimental period, blood samples were collected 1 is suspension 0 . 5 ml o f a lOOx 106/ml ours post prandially at 08.00 for three consecutiv ~ s i o nof boiled baker's yeast in phc~sphate aYs (fed samples). Feed was withheld for five da) red saline was added. The suspensioIn was nd s a m ~ l e were s collected at 40.64, 88, 112 and 13 r n l x e u on a vortex and incubated at 37OC. Aft1tr V, n L, I n the fir! ours post r (fasted s jt 5, 10 and 20 minutes the mixture was vortex,ed and samples were taken with a Pasteur pipette. !Smears a s : Departmcnt or Vcfterinary Clinical StudieS, an, SN7 0WC) Saskatchewan, Saskatoon Saska~chcw were..- made o n microscope slides, stainec1 with --..-"" ied Wright's strain and tht: percent;3ge of modlf Pracnt addrC ~ S .c ~ ar~rish u Embassy, PO Box 801. Khanoun1, hdan. neutrc)phils cont aining yea.st countedI at each ti)me. ....I-..*-:A:-
rn,radermal
. . A -
:f ,f
.
..
! - .
--
.
.
.
Statistical analysis 1was perform e d usinl;a pockett lculator . - . w. i t h statisti - ics modulc:(TI. 59 F'rogramm.11ecalculator; I exas Instruments). I nere was n o ~nificant difference between fed day results for :her haemograms o r neutrlophil phagocytosis ,tectable with theI two-tailled paired t test. c-- L I I eref fore, mean valucb, IUI ~ ~ a e m o g r a m variables and neutrophil phagocytosis, were calculatLed for fecI da.ys and were used in subsequent compaIkens wittI fa:sted days using the two-tailed paired t test. At the first skin test the control and experimental groups of horses had similar sk;in test r eictions foir both the 30 and 240 minutes respons,es ( t testt P>O.l). For each group of norses significani anges in the skin test respionse to s;ubsequenl allenges u'as compared using two way 1Inova anc uncan's ne:w multipl e range test (Steele ;and Torrie 160).
-
.a"
P
..
7-
-r
..
. ....
-8
I
T
T
T
suits
Total mat ure and ~ I I I I I I ~ L U I~leutrophil C counts dc jt change significantly with fasting (Fig 1). D u r i n ~ ie days of total calorific deprivation blooc rnphocyte counts fell by one third and ther covered somewhat during the refeeding perioc 72 ig 2). 1 Neutroph il phagoc! rtosis decllines durir~g fasting sfeeding Hours c,f food depri!lation Fig 3 the time cours;e of the plhagocytic Iresponse i!j .-..-. , .L--. __,mparearor iea, rive aay iooa aeprlveu anu rnrrc rlcl L: local Diooa lvmDnocvte counrs. meanzstanaara aevl iifferent day refed horses. Day to day changes in the 2() five horries. Values m:arked with arI asterisk are! :0.05 using 1'he paired I te minute phagocytic index are shown in Fig 4. During: from fecI values at P< fasting neutrophil phagocytosis fell significantl) f-3m a mean of 97 per cent in fed horses to 78 pel nt, after five days of food depr IThe skin test res]ponse t o histamine:, did no ttests withii ~ d e r g osignificant ch anges bet! ween. skin I . ther the experimental or control horses (Anov:
.
- 3 fl
3
>.--?..->
--J
11
I
-
[ ' I F I
A X Horses Five dayfed fasted
ree day refec
I
-
1
4
1
1
1
1
88 112 136 24 48
1
72
-\
w n of food
;of
deprivation
refeedin
IG 1: Total blood neutrophil (mature a1nd immature]I counts in fi! ~rses,data are expressed as meanestan dard dwiation . None of tt "t..... ..-:~luesare significantly different from f hA warus> urlllll palrca ... r,
..
'"0
XxX
d
V
V
Y
5
10
20
Time (minutes) C l U 5 : I I Ime course of the in vitro phagocytic response in four horses. Data arc:expressed a! ~darddeviation
371
Host defence in food deprived horses
second skin test in the control group using the f test, p = 0.01 i
ussion
..
. .. . .
.
Peripheral blooa lyrnpnocyfe counts re11 aurtng total caloric deprivation. Similar observations have been made in dogs (Dionigi et al 1977) and malnourished sick people. Rats fed protein dleficient cells in diets have decreased numbers of lymphoid ,.--. spleen and thymus (McEarlane andI Hamid 15113). ~n this study blood neutro]phi1 concc:ntration 1l ~ a snot affected by fasting. Th is is simiilar to re sults in *--,\ . ~. . healthy people (Palmblaa IYIO), m conrrasr caloric :reases blo deprivation dec od neutrophil count in dogs (Dionigi ~ et al 1977) ~ Decreased neutrophil s not seer phagocytosis i: I in healthy fasted people n7-n VL pL.dple with protein calo-;a-n1'L1b L l l a l .he2 eta1 17 1 tion (Seth and Chandra 1972) but did (:xcur in study along with a decrease in the Arthus mse. Decreased concentrations of comldement . .et. a1 proteins are seen in calorie deprived dogs (Dion~g~ 1977). There are decreases in a variety of comvlement proteins in protein-calorie malnourished people (Smythe et al 1971, Sirisinha et al 1973). Similar reductions in complement in horses could explain changes in neutrophil function and Arthus reactivity observed in this study. Complement is involve!din the Arthus reaction and may assist in the opsonis:ation of yeast by neutrolphils. We have doc:umented marked dc:creases in several arms of the ho!;t defence system following shc)rt term -- -,-I-:1L a :--I food de~rivatiurr. I 111srlab I I I I ~ U I L ~ I Il~~t~lications L horse W I:Ifare. P eriods of electivc: food ivation d uring tra nsport or for vet erinary oses shor~ l dbe kc:pt as short as p~ossible. .exia commonly accompanies illness in r i r b horse:s and ef forts to iimprove 1'ood intake may bolster host derfence.
.
.
. . 1
-
.
1
2
noun of refeedinc
"-.-T
leprivation
~-
IFIG 4: Neut rophil phagc Imean* standl , . . . ~n . ,lour . norses. values marked!O.wminutes. .t h. an asterisk oevtarlon are sign... nt from fed v,slues at P(O.05 using the paired t test
.
Test horses
- mtrol horses
1
A . *
... ,.
II.1".
This work was supported in part by the Philsdelphia Foundation and Biomedical Rpcparrh No SO7 RR 5464-15. - ort Grant USPHS - - -
Rec,eivedforp
u .
allergen -L-Il---
8 , -
IC
FIG 5: Conlparison of the Arthur response iln control and experimental horses. Cc~ntrol horsc:s were fod through01ut. Experimental nones wcre fed at first a llergen - . tor - challi .:nge, fasled. * ,1br 'our davs at the second challenge and reled ltve days at rne rnlro ere were st% horrcs in both experimental and control
-
...
January 2
References
Third
First allergen
I
DIONIGI, R., ZONIA. A., UOMINIUNI, L., CiNtS. b. (e RAl-LABIO, A . (1977) Annals of Surgery 185.467-474 -. -. HALL.IWELL, R.E.W., FLEISCHMAN, J.B., MACKAI'-SMITH, M., BEECH. J. & GUNSON. D. (1979) Journal of the American Vertprinary Medicine Association 174. 277-281 LAW. D.K., DUDRICK, S.J. & A B W U , N.I. (1974).Annals of c. Jurrerv 179. 16%-173 McFA & HAMID, Imn 153-161
American Jo
ELD, D.S. & ACLAN! srinary Resea,rch 41,899-9
\LMBLAD,
rndinavian
~ Y L O R , J.I
-.- --.
Jc)urnal oJ Hat
1)
'
LI I-LLO
~ A N C H E Z , A,, K L L ~ E K , J . L . , L A U , ti.>., I A ~ I A U , r.1 ., WILLARD, R.E., McMILLAN, P.J., CHO, S.Y., MAGIE, A.R L. ®ISTER, U.D. (1973) American JournaloJClinicalNulrilion 26, 1180-1184 CHANDRA, R.K. (1972) Archives of Dkemes in
ID, R., E D E L M A N , R . , S I R I S 1 N H A , S. ILSON. R.Ei. (1973) Lance1 i, CHA RUPATANP 1016- 1020 JLVl l 1 rIE, P.M., SCHONLAND, M., BERETON-STILE?3. U.U.. ' COO VADI, H.h4 . . GRACE1, H.J.. L(3ENING. \N.E.K., MAFIOYANE, A,,, PARENT. IM.A. & VOS , G.H. (1971) Lancet ii, 935)-944 ocedures STEEL1E, R.G.D. &TORRIE. J.H. (1960)Prir1,.,.,,~.... o f Statistics. New York, McGraw-Hill. p 107 0. ' " T T
...