REQUIREMENTS FOB VITAMIN C
AS AFFECTED BY EXERCISE
by
PHYLLIS MAX CARPEHTER
A THESIS
submitted to the
STATE COLLEGE
in partial fulfillment of
the requirements for the
degree of
MASTER OF ARTS
APPROVED:
Associated/Professor of Poods end Nutrition
In Charge of Major
<i'*iiiii<iiMiiii mfrmov.^mmmmmmmmm.m u+mmwm*>m*m*mit*iia,m^**m*+miimi0**¥+'m
of Depcrtmont of Foods and Nutrition
if«Mn^nnvn«<Mw^iM«^«««Mn|w#iM|Milp*'
CheirmRn of School Graduate (eommlttec
MPW|l*»' *'■ Piilliiij'i qatmim*****
Ch&lrraen of St&t^Xollege Graduate Council
ACKNOWLEDGMENTS
The writer wishee to express her gratitude and
ppprecietlon for the edviee and kind esBistence given to
her by Cr, gfergaret L. Fincke, ^asociat© Profeeaor of
Foods and Nutrition, during the course of this investigation.
She would also like to acknowledge the loyal cooperation of her three exporimentel subjects and the Interest
and support received from B^s. Jessamine C# Williams,
Head of the Department of Foods and Nutrition.
TABLE OF CONTENTS
CHAPTER |.
INTRODUCTION
1
Historlcel Background ............
Chemical Properties of Ascorbic Acid .....
Phyalology of Ascotblc Acid .........
Methods of Assay ...............
Methods of Determining Requirements . . . . .
Evaluation of Methods for Determining
Nutritional State and Requirements . . . .
Plasma Levels
Requirements .................
Factors Affecting Requirement ........
1
3
4
14
16
25
27
30
35
CHAPTER XI.
PURPOSE OF •fHIS IKVES^IOATIOR ....
4l
CHAPfBR III.
BXPSRIMEHfAL ............
42
General Procedure of Experiment .......
Analysis of Foods Used in Diet ........
Procedure for Determining Plasma Ascorbic
Acid Level ...........
Standardization of Dye ............
Stenderdisetlon of Sodium Thiosulfate ....
Solutions ..................
Description of Subjects .......... .
42
45
RESUIifS MX) DISC0SSIOH
GRAPfSR IV.
CHAPESR V.
COnGLOSIOKS
BIBLIOGRAPHY
46
47
49
49
50
.......
53
.............
57
LIST OF TAM1E8
fftbl©
I.
II.
III.
IV.
V.
Daily Intake of Vitamin C Requirecl to
MointRin Tissue Saturation 40 Reported
by Verious Experimenters . . ......
33
Ascorbic Acid Content of Poods Used in
Experimental Diet ............
43
Daily Plesma Ascorbic Acid Values,
Subject AR ........*••....
§3
Daily Plasma Ascorbic Mid Values,
Subject LR ...............
§9
Dflily Plasma Ascorbic Mid Values,
Subject KD ...............
60
REQUIREMEBTS FOR VITAMIN 0
AS AFFECTED BY EXERCISE
CHAPTER I
INTRODUCTION
Historical Baokgyotmel
Scurvy, a vitamin 6 deficiency disease, has been
known In history aa a plague, infesting armies, navies,
and besieged towns.
It was particularly common among
the cruspders, on the voyages of oxplorstion of the sixteenth century, among the colonists of th© northern pert
of Amerlem, and throughout ©11 history in the northern
parts of Europe and Acis.
The Greek, Roman, and Arabian
writers do not seem to have been acquainted with the
disease (55).
It hes been known for over 300 years that fresh
vegetables are a potent remedy for scurvy, but it was
not known until the early part of the twentieth century
that scurvy 1$ an avltamlnosis (36).
In that same period,
it was discovered by Bertenstein and, later, by Hoist
and Frolich, that the guinea pig could acquire scurvy*
that discovery opened th© way for aoiae valuable experi*
mentation relative to the nature of vitamin C deficiency.
Between 19l8 and 1925, Zllva was able to obtain
almost pure antiscorbutic vitamin from lemon juice*
He
was able also to determine the basic properties, such as
molecular compoeition (empirical formula CgHgOg), its
2
resemblBijce to the hexoscs, and its instability to
oxygen, e ape c I filly in e.lkellne solution.
In 1938 Szent Qyovgyl isclatea from rdrenBl glancis,
fvom oranges, end from cabbage, a strongly reducing substance vhiefe ho edited "^©awonio aeii.'*
la 193%» tfee
identity of vit©iBia G wttfe jtoettt %©r$^i*8 "feeaaspoaie
a^ld" coad ^itH ^il^a^s (,vedtteltts ftoetw* m& ii0@©yoi?©d
by wsflpioae .gpo'ape' of ttOPkeFs, iiaao}.^ Hfeiigli sad Kia®*
Sti»b9l.|r aad Sa©at -Ct^orgyt^ aad fillmaaa CB6).
P&PQTB
^Ao fl»9t
bf !feag^ ©ad Siag woap© baaod maiai.^ ©a tJi© ioola*
tioa of ttosl* firiat ©atiseorbutio ersratals ia 1931.
MeoMiag to Sing (61)*
Tboae e^yetalc, obt&laed from lonoac,
correeponueci ia t^pe, aeltiag |>oiat,. solubility, MtJ?ati©a yala© ©gaiadt ©sidi^iag
and reducing agents, rotetion, electrical
trassferonce, aad G -- 1 coabustio.i, with
th© "hexuronic aoid'' which Sseat ^yofgyi
h^d isolated as a tiaeuc recpiz'Gtory tmkQX
from cebbsgee, oranges, and adrenal glcnds,
at ®asabrldg© 0aiiireJ?©it|r ia 1^08*
■Zhevo seomad to ba no reasonable basis for queeticfilag the ideatity of tlie vitoiain after it taras deraoastrated
^hat Bte&dall*6 iad©p©ad©at ©xtraot frsaa ad^aaal glaads
aM by ^a ©atlrely different pveeedove liad tfe© staa©
antiscorbutic value ad the product of Waugh end King.
Eeichstein aad Heworth announcea the first successful
synthesis of ascorbic acid ia 1933.
3
Chemlcel Properties of Ascorbic Acid
Vitamin € li®$ aa acid fresottoa, %%t& |>rop0j?ty beiag
doe to an enollc hydrogen (59).
'Tbe most algnificent
reectione ere those which involve oxidation and reduction.
fixe oxidstloa of aseofrbic aoid 1& gteatXy ©ffectod by
verlations in pH end by the presence of catalysts, euch
as cupr-ic, ferric, raorcuric, Gilvcr, and mongar.ccc ions.
T^e vitaai© is j?©v©l?0lbiy oxtdiaed by a g»eet many
rcfigents such as methylene blue, qulnones, the 1' dophenol
dyos, hydrogen peroxide, and iodine.
In cqueous solutions,,
the mt© of a$j?©bi@ ©xldatloa %■$ increased by exposure to
light and by the pi'ceenee of the hemochromogens.
The
oxidized form of the vltrain, dehydroescorbic acid, 18
falply atabl© la toUtatloia belot? pH^, bat la more alkaline
solution, another strong reducing substance forms, which
on farther oxidation gives rise to oxalic acid and
1-threonlo acid*
Reduction of dehydroascorbic add can
be brought about by hydrogen culphido, eysteina, acid the
"fixed - SB" groups of p^©teln0*
It %m boon found that
glutpthiojuO in the tlcimes may mt as a protective mechanism against oxidation of meoojpbic ^dd by <a©pp©i>»
Accord-
ing to Becker, ascorbic acid is considered the ooonzjme
for glntathione oxidation {6)*
f^o of the eneyiaes which
©xldla© and reduce ascorbic add respectively, ©an b©
separated froa cabbage juice; the reductase has been found
4
to be much more labile th&U the oxldase (22).
Phrfrioiogy of Afeorhio Aeia
When aecorbic acid ie withheld from the organism,
the main changes that occur are in the intercellulfir substances surrounding the cello which stcrt from the
mesenchymal or middle Ipyer of the embryo (24).
normally
the flbroblast, or the basic cell of fibrous tissue,
forms email fibrils in an pmorphoue ground eubstEnce, the
fibrils being gathered together Into wavy bends of collagen
of1 connective tissue.
In normal growth, the fibrila
Appear to he cemented together by a translucent ground
aubstance; It Is this formation of intercellular materials
which is probebly controlled by vitamin C
In ocorbutlc
guinea pigs, the fibrobleets and the ground eubatance ®,re
present, but the fibrils and bands of eoll&gen do not
eppepr; when the animals are treated with sufficient vitamin
C, the collegenous material appears in eighteen hours.
The formation of the Intercellular substance of teeth
(dentin) end of bone (osteold tissue) can he controlled
in the same vay, by supplying or withholding the vitomin.
The bony lesions In vitamin C deficiency are most
eommon at the costochondrcl JunotIons, the distal and
proximal ends of the long hone of the thigh, and the proximal end of the tibia and the wrist (26).
It appears that,
when vitamin C Is not present, the osteoblasts return to
their primitive form (flhrohlasts) and try to form a
fibrous, rather thm ?- bony, connoetIon, between the
cpiphyuis find aicnhyuls. This abnormal {son© is called
the ^gerufltwMrtc* «r "ftpiaevwie ai®i?rot(r,." ■ir&tcti id tfei©
i!k 4ofiei©©©ir 0? vit-Qsaifr 6 i»©smiti. i<i ©tenges ia th©
te©t& ond gums (ii#S5*)* ^©s*© ai»e e3i®3ag©8 in the pulp
a&<3 &^ntin of tho teoth, nna 'virinflaiffi^tioa of tho gome
Pdealtd* ttta©!?' soytft of t&© bod^ Qtiiefe may b© sff©ote4
^y asoopbie a* id 46£l«leitty «po tla® ©f©s (15), tfe© i©si©
adwov ($3)* «®& gla»fiai©r (83) «ttd saa^el® tldoqe-{lO)«
I3©et leaioas of soyo?^ osia b© traced to tho loablllty of
tu© aoeoiudg^aal oettt to fo^a. isappopliag oabsKfiaeQ* ih©
typitioi issioa of ©#©&tir is tu© pe^ifoilioulap p©t©©tei#i
h©a©i»Pb®@©* fh© p@t@<3iiia<D ay© most eoaii©® om tti© logs ©F
^©?0 tbef© is prtennvpe m th© oafiliftPiee.
Cramkm, Lund, cna Bill (31) rceaatly co^rlbuteel a,
vfeintebl© e^aiF ia vltami* Q deflei^tMijr. ftoeiir 8©b|©®t,
toaftdon, tsra© o© a diet f*©© fpon ir©g©t&bi©si t tmtt®, end
Okillc fflp sbout 6ix Eioatlis. At tikm ©M of 4l d^f9* ftio ■
plftgiiaia %9V9X of ©sdofbi® ©eld' »a» eevo* $h© a.aoo»bl© aeid
content of tfce wMtoi-oell plat©l©t blood fyaotlo^ foil to
■aep© &ft©p. 83' d©js* §a th© 13^ti^ day, Ssnall pwlfolll^n*
Iflup papaleQ appeefltod o9©x» tho battodtes and baok^ of tlie
ealtr©©.. !J?h©0© papal©© fcliey e©nsld©r©d to b© tike flffet
slga's of mmvy* ?©s»lf©lifoi|ilik5p ^©iio^3?li§ig©0 ^.ppemfed
sft©f» l6l days., %©a tb© 8'«b5.©#t va© im tb© deflelent
'6
state, c>n incision was made across tho lurabas? region of
the beck.
This wound foiled to fcecl until the Recorbl©
a©ld 8«ppiom©nt was givea*
¥iiifl©ia aa<a Srvtia (47)
Rdvise thet t|i® plasma escorbic mi4 and the serum protela
levels of patients fee brought to ftOJPm&l befor© ft^gery*
men vitemiD G rioh foods ere eKCluded from the dlot,
& vague condition of general poor health msy result (69),
the state often being eceempenied by anemia (^,66,69).
A deficiency m&f fileo be Bccomppnied by a loworeci reoietrnce.
King end Menten (60) found &, definite loweri^ of
the reeiBtenco of vitrnin 0 deficient guinee pigs to liajecttoas of dlphthei?!^ toxls.
Florl (3$) demonstj»at©<i thst
ascorbic acid alone or with extract of adrenal cortex hpd
soffle detoxlfTlag setloh is vitro ftgalMt diphtheria.
It has been suggested that a lack of edequete vitDJain
C may be the oa&se of hyperaenaitlvenese to certain foods,
particularly as it occurs la iBfaney (91»li2}*
There h^s been some evidence presented (82) that a
vltsmin C deftclenej say lead to respiratory ahd other
infectlone.
The effect of ascorbic acid deficiency on the reeiotrnce raechanisrae of the body is hot known.
It is known
that there is eoiae relationship beowoeii asoorblo acid and
blood complement (68).
It has been found that there is
a drop in the complement tlter of the scorbutic guinea
pig and that the value rises when vitwain © is adaiinistered*
7
aai QbeppwA {7Q,®2) torn® fhBt with vt%m±ti © QotMoMf
tfo®ve 1.9 6 X09& i© ipeie^t> nhioti is oafetapfcntftd Ijy AB.
tiomtisY, .iJ©a^®a,' aa^ tins Cl^) ®a^© easnteaisiv^ liietol©*
aid of iitv&t nlt&etie m& f*t st&i&iag n&tefialo epsltei
ilredfely t© fke tiisstt©©* ffeej' FottttAsttnt t&© siiv®^
t/ep© oa a vita^lia C?' fre© ^i^t*.
I© tsli© Sfeo^toutie guises
pigs, tbey »©t©i tls© <lliia|KP9ei«Me «f .f«t ntdialag •
mateyi&t ftoft of ^oobSL^.^ftfa^tiird ©belest©!?©! fi?©© ^©
(aflpoiiai. do^tsosc* Sbef $,%$& foiind ffet^f ddgenei^a^ltMb itt
pcrto of the nyocLirciimn -cmi lit ol:oIett\I aut;clc as veil
m $&%%$ tnftltPBtto^t m^ t&btif 30ge8e*&tl0ft of tte li^ea?
aM ct)n©':>inico ccngostica of the iivc'rj tmd homol-'^hf.ecE.
MiiTRlrerfli (73) found that In guinea plga i'ed on &
diet deficient in vitcnin C, fli@ pigraGrit Gr,crotiiio J.'^KCtio^ of ^&e iiws» snd fc&e o-speoitr of Kto liv©x» fos? da*
to'iifyine ii@€itti!i GGntonlaun fend iiiuolo wore ieere&aeS*
•Jbe oxidafci^o rerotionc ci* eaoorbiC acid stggo'St that
the vitsolft se^" pi&5r ©a iiap©^t.©at patt ia ti&tttse i»'©spiip&*
ticn. Keliie and Zllva rop&Ptoil tiict EriTiaoii found ?in
8
uptake of oxygen by liver sliceB of scorbutic animals
wben vitamin 0 vee added (56).
Kellie end Zilva were
able to repeat the experiment and found that the eddition of ascorbic eeld to liver tissue increased the surviving respiration even when the animals were not scorbutic.
They also found that the increase in respiration
was less in animals on a normal diet than in anim&ls on
& diet restricted in calories or in ascorbic acid.
In
further experiment;), they found that the surviving
respiration of the liver tissue of 95 pev cent ©f their
animals on a diet restricted in calories and 5% per cent
of those on a normal diet was increased by the addition
of ascorbic acid.
The great reduction of oxygen uptake
which was brought about by phloridzin, inhibitor of
phosphorylation of carbohydrate, suggests a connection
of ascorbic acid with any one of the stages of carbohydrate catabolism following the
tep of phosphorylation*
Stewart and Learmonth (93) found that ascorbic acid
injected into cats which had been bled to 50 per cent
of their original blood volume would, In some eases,
prolong their lives.
They suggested that the action may
have been due to the increased supply of oxygen to the
tissues by ascorbic acid and even suggested a possible
practical application of this phenomenon to human sub*
jects during war time.
9
Other biochemical funetione of e corbie scle
have been auggested.
Robertson, Ropes, and Bsuor
(83,84) found that ascorbic mId would reduce the viscosity of mucin in the presence of hydrogen peroxide*
With further experimentation, they found that it acted
on polyaaccheridos auch ae starch, pectin, flexseed,
mucilage, and the polysnccherides of synovial mucin end
cartilpge and that it destroyed the capsuloo of various
types of pneumoGocci.
Leter thoy brought ©bout the
in vitro dephoaphorylation of /^-gljreei'o^hoophfete hf
aecorbic ecid and hydrogen peroxide.
They suggested
that a possible function of ascorbic acid maj be the
degradation of mucina ^nd polyssccharides in physiological
and ppthological proceasee.
Itoggle tind %nd (77) suggested that ascorbic acid
mcy have a role in protein aynthesis when they found
that the ratio of insoluble to soluble nitrogen in the
leaves of cereal grade increased with increasing amouiite
of ascorbic acid.
It has been found that ascorbic acid has some function In protein metabolism*
In 1939, Levine, Harpies,
end Gordon (6k) found that certain tyrosyl compounds
(1-p-hydroxyphenylactic acid and a keto acid which they
believed to be p-hydroxyphenylpyruvic acid) were excreted
by premature infant B.
They found that the feeding of the
aromatic aralno acids to these infants would increase the
10
At 6bo«t tfcft sass' tSisdf S©4l0#k and Siib©»©:t©im
tof f#©4i®g ritenitt 0 4te'#ieient gtiia©s plgi I^POPIB^ ©^
feoAtog of tte vitamia*
m&immi (88) f»aaa tint fecnogeatiale &©ia* alpha, koto
g«ti»«|ft pig© ^h©^ ©itfeep l^sKkdaylaUitfttfi ©^ ^m^ipf^mle
a@ii was giir«ak Wfeea ©m© of tfe© a©t^ollt@©, jMtytfsoxjr*
pfee^flp^^tri© ^©idj wi.8 gltr@m^ tfe© vltttailtt WB fouai to
h&tf# tltti© ©s» .ao effeot m the •iMWoeAtttg oetaboliteo* ■
ftras %lkl0 WWijr oowil^Utoa tlttt tfte ^©iat #f attaek of tSi©
titdaift ©»t ©iai»© ddldt notftbelitsBi i» oenfieod'to tins, early
•tage»#
•ey«r (IS) ira$ ablo t« pyoiae© th© laaetl^tio^ of
»ityo> H@ ofetfei®©4 up to a Si p®? eeiit yt©!^ ©f ©saaoaife
t»l0E ftis» i»£6 bufebi©€ tfapoegfe a. mtiem& ftolmtioa of.
ceeorbic f citi end eomc rminoa. He G^BC t'cnmd a (iecreased
©2&cs?©fcl©m of osg^otttaltio by Aoge vtaon t&^y we»« give®
'82 to %O0 xag. of asoorblo aeii.
11
Bourne (14) nee suggested &%ilt another function
of asooroic acid.
Be pointe out tliat ascorbic acid ie
located in high concent^ciiion la the region ®f the Goigi
apparatus of biologically active cells mM tli&t the
Golgi cppai^tue ig yecogniiied as the syn^I'etiic centosj
of the cell.
He concludes that ©sCGrbie acia prei>ably
has some function in the s/utheeis of chemical tjuhataiioesj
lie gives at the most likely explotifetioii that the presence
of escorbic aoid fotmm Im %&». ftT&t&ofeie elates? of %hs> oel'i
pj'oaiaeit ft©® %Qi®$ m0%®'b®%l&Q& as deea as fosis®^*
f|i9'-pela.tioiidtilp of ©se@pMe m±& to opeelfltt toFffioa©s a&d. tfttd^adfi has beea itMi^fi -in a^a© l^©^^®?!©®*
^os»® is ©©a© ©;t?id©m©<s' ^Siat ®1©' *&fc6&tfe h&@ ^s aati*
fetjirolj^opi© a©'%l©a, hn% tli® r^lati^aeii^ iA9 wit b©©a
-ftigift} e»d-Siiig.f93) ?otts«S t&at tfe© g'la@@s# feotaparoe
of giiiaea pigs ^m defiaitsly 1L#^@TO^ bj p^ogf^ssiv©
®86©3?l>i<? e^lS :i©pI©tiosj»
p©lati0iaaiip b©tti@©ii g$tio&bl$ a<si€ aM fott? ©a^fsa®©, %wo
fefirot^ie am<l t^r© feapimton'y* #«P tito stvfl^ of tl©
ji|rdi»©l^ti.e- amsiiita©©, tlba tlsfifioo it$e4. x^oipe tftentoted with
of tto s?e©pi:sa,to^sr onts^o^t)
WJP©
s^Mio^i % aoefiiKPlag ttoe
12
oxygen or hydrogen uptake cf certain tissued in a. unit,
time.
In this study, they found that the liver estersse
activity of their scorbutic guinea pigs vas one-third
the activity of the normal animals.
They found that
phoaphatese activity of intestinal raucoae and kidney cortex showed a moderate decrease in scurvy.
The activities
of the two respiratory enzymes es meseured by this
method were definitely lower in the scorbutic animals*
The pctivity of succinic dehydrogenase in scorbutic
heart tisane appeared to he SO per cent lees than that
of normal tissue and 65 per cent lese in scorbutic
skeletal muscle then of normal skeletal muscle.
The
activity of cytochrome oxidaae in scorbutic heart and
skeletal rauecle was about 45 per cent lover than that
of the normal tissue.
Harrer and King (42) suggested that ascorbic acid
may he important in cellular physiology aft a regulating
and proteotlve agent,
they write ai followst
"By
protecting active groups such as R * SB (1 being a
protein), relatively small cmounts of ascorbic sold
might aid in the regulation of major hydrolytle and
respiratory systeas In the tissues without entering
directly into the reactions concerned."
They point out
the apparent paradox that although scurvy is eccomp&nied
13
by total inc^OQaoa body ^©apl^tslom esd Ind'eaoed Fed*
pirotion of tissue ollces, there t$ & distinct decreftse
ia aotiv&ty of ttro- of t&e s»s.j©? t?'®®pimtQ$f m%fm&* ■
- ^e vote of seootbi^ aoii ia tfcd sietaboiio pffoaesaes
of plftirSo and aaim&lp Is not oloB^ly understood. ' Thove
le pi»s0ti^all,f »o asco^tol© aei^ to ^© found ta 4>y seois,
trat tfe© vitdtt&ft afpe^p0 vlievo ti© 0fi?otitiag. pvommm
&?e ^pes?®ati|' laitint©^* ia § few feoit^s afte» 'tite «©©d
is a©i'jBt©a©d* 11 is foisaiS iii Mgh e@ii©©Bt2?©tl©ms la
%!>@ a^^lvdty ii»0ulag' pi^ts ©f tla© hi^Si©^ p!Uat9^ aM ia
all f5P0.3& p>©©® ldAire«« la. the mijml ofrgmtm ©seos?^!©
a®14 is forma ia t^a greatest aoncsatratloao la the
tldftttee of g3P©et©fit detivitiri tUe pituitel^ boiy geae*filly Si^s th© ^©atdet c©i&@©at2Patt©a a®^# ia ^©a^ea^iag
©fi©^,,.' tli® w&ptte lat©©!,,. @4j?©aal ^©ftex,. jroiaig tlipiu*,
livep, @aS bi'aln. The presence of the vitmain ia. tissues
of hl^hdfirt metaboli© aotivitf ia€itiat©i thst it nftp have
sa ©©©©attel aetabdlio I?©1©> Hi$g (6i) states $ "Slie
high oonceittrctlC'sa of socoi^bie ecid la. glsadular tissue a
ia€i©st©© * 4»$p94 ©f ©o^TOlatioa »ith th© $®mp&% #®t©
@f tt&tdholKust aiiftlQgotts to th© ^©latioaship ia pl©at0♦,*
Kei^om'inri (52) fouM that ia blecfl feept la ^a ctmcsphere
©f :altF©g©a> th© plaeia©, ■©so.oi'biO' eoii ©at©i*©4 th© ©ella
pt .room or body temperature but not at low tempes-'aturee
(7 d©gj»©©@ ©©atigs»M©)» ■ FiPes! this h© ©oaoia^et th&t th@'
14
transfer Into the cells Is probably aaBoclcted with some
metabolic process.
Methods of Assay
The eerller assays for socorblc ecld were mede
with the uae of the gvilnes pig, both by the preventive
and curative methods.
Another method that has proved to
be very useful la the "guinea pig tooth method0 suggested
by Kd'Jev* la whleh the degree ©f aeurvy pyeeeat 1$
d&te&nlfked .hy #hii.iigeS; 1® the st^^tos*© of the (tsfttlm ©f
the. t©®th»
I'llLlsieii.e p Sissiohj suS Hii'-seh ftJPSt developed ©
iidthodt, JP©5?' .(Set-^urolRlu^ .the ^vese&o© of s?©<Slui©e^ fts^ofbl©
aold iB; hlo©^?-
^helip. ffieth©^ wafc hase^i ©& the tlt^atloa
of a t^lshlo^sietld fe<s:i€: tl^tJP^te, of blood ^l^Sffife Hlth
■ the d^"-® ,@ ,6, di^hio^^hetiol .Itidlopheteoi*
IpEieri©; «ttd vaa l<s&eleB, ©tig^ested. Feduciag, th©
as^oFblp aold ^etesat.iin hXo©d plessa vifeh hytTO^en,.
9ttlf Ide. end. ^eeifitgitlag': lateyferlBg' s^ucisijl' .^^biatsBces
■«aeh. ^©'.itsyateim©,, with seiwivl* s^et&t® fS®)*. .fhls
sjothod.-hs'e-^yev^d t® he y^th^j? i[ii@e3?1lal9t^ sts It Is
a©d©.s.8$jp.y to ^©©©ve the hirdpogeh sisl.fl«l® c©a®letely tilth
alt&©'g©a !&■ ©fdei? t© pi'eireat ■ e^©©0i$ i?©dis©tl©a' ©f the dye*
Wmm&w :m& Aht have ©©dlfled the ©Hglh&i dye !>»»©•■
6©dii9e. ©ad have adapted It t© the ise® of veK'y small
©aaoasts ©f. feldtd-*.
S'hey "$tigge$t©d using addton. twogatote
@M!&t, lat©3?j .raetapho^ph©^© a>©ld». &# pi?©t©lsi pi?®©i^l*
te»t» (>®K
IS
potfeaslui^i cya&ide end potaoeium ox&l&te to prevent the
c^idatioa of eccorblc acid In drawn blood samples (79)Other vorlcei^ have f^m^d that there is
BO
gro&te? loss
©f iaseo^M© mi& vhm pote^eitni ©f&tal#6 ie not' fe«ld©«J
and that ooaie lots cf the
CG!^
Ve®±m& oth®® reageatg
tou-i to dacolc^l^e t^®
&B&Q
ho&n aaggeeted for as*
e®i?tei® acid ajaalfsiSi -among «lai©|3t a*d f©rs»oef®ai^®,
pMos^feotungatl© acii^ ffi®tli^S.eB© bl-^e, sad flioafiiosolfMio
p^ovdd t® b© mot ©ati^ei^ saiilsfs@toJ?f*
A epeets^*
©copi© ae-tkot <md & prsteoiur© b&«&d om t^o «so of «tid&*
tioa*r©<lwttom
tie
QSQ
*IBPW-O MV©
b@©ii doggested*
800 smgg©S!te<l
of SHo ^oaott®a botueea s^Ilim© oaotate aad
f«^fa£»sli ^M©^ i# foHE®i »©« 68«oybl« a@ii 01& f&a&tYs^io^s 3pot©iiti^80t^le aoifttedd fe&vo b©©B t^iei,
btt* ajS^il so^.p tfeofi:© to© b@©*i vor? Uatl*©* ia wtmlHon
bmmm- of ^iftiEg p©t©stt4l'# m® MAt&ttmt ©mi-points.
B6s^i6# ^psom*. m& Wang (44) feave reoe^tlsr iat^oi^©©i
a ®0tla0i for dft- $le$t2flttet»to ©lalftlt witli tSi© us@ of
a flatimwB et©et3P#a© ©mtot «lt& awS'ftttSfy* fho ©©tfeoi
taa^
^IPOV©
t© b© a us©fmi on© f©# vdattaa saatjreia* from
16
MIndlin and Butler (/2) imve adapted the aic^o
dye met bod to the photoelectric e&lex'imeter.
Of the
meny mic^o iaetliofia tried by various workers, the indophenol titrBtion method ae modified by Farrcer ana /ibt
and tit© colorimetrlc metiioa of Mindlin aai Ittlet Mve
been prefeiTped fey the greatest noiahe^ of «wk»#&»
Methods of Deterralnlng Requireaecte
the fir at attempts at messuritig hxmzu tequii-emente vere raatie i;y C^tiiliu in Bt^QdOE la 1931 *
li&
method way to tlad the saallsdt caoaat; of o^&mg® juieo
whie& trottlA ^©x^eet © 1099906 e&pil^tey jfc&%&$6M®t a
condltloa vhioh
WGS
^evoslod ijy goiocaiao a^poari@g tihen
pmarnvQ va* applied to th» as?a (?7#-38»39)*
S^io method
has beoEi ps?tl7 ivplacoci ay a *3@tk®i iiit-'odaGoa %
Dallclorf, vhoreby aagativo p-?ossii^c is appliec, t^ aa
©pea of the avm and tke tiu^aba^ sf p^toaliiao oomt^i (9^}«
Ha-^y «03Pkefs sin®® <|$t&ll&*e ®xf©3?lia®ats. to^o pel?*
formed have found no
GQWOISXIOU
hQtii$®m o^pillc.^y
i»e®l*ta®o© aai astritiohel et&te (il3*7S,4^#as}»
Ae«o»d-
ing to (iiJthliii, the ^e&fioh %hat th©@@ uom-si'e Mave found
no sucii coiU'elatifcu t& thrs'c the ^etho^ is mot Gpo^xfis
when jpl&sqi© levels are Kuck above C.l pg pm 100 ml anfi
th&t ii* the oouuttles v^iie^o tiiosc o^pstieciA'ic ha.vc I/^en
eavsled oiit. (except is Sweelea), the ©wi'a.g© &i®% ia e©m»
pai?ativel>' floh in vliesiiii C, af>d plaism '^ilnoa
GG
low
17
as thet are seldoni found.
Dalldorf hse made extended
studies on tit® method, using his negative pressure
technique, and ia of the opinion that the method will
^ive dependsble inforraetion on the presence or absence
of vitamin $ deficiency but does not ahow the degree Of
defioiency.
Dslldorf has found th&t the method yill
give f&tti®%f neg&tlve results when severe anemia is
present.
Sloan points out thet the tHioMeat of the
skin and the position of the arm also affect the
There is some evidence now that lowered capillary
resistance as shown by these teat® may be Sue to the
leek of vitsmin P (65).
Subjects of Levcowich and
B«?tcholder had larger peteehiae when on a %W vitemin C
diet supplemented iiitfe 4& to 120 mg of pa*© ©soofrbi©
acid th&n they did when
OR
a freely chosen diet*.
In 1937, Hotter introduced © direct teat for deter*
mining nutritionfel state (87).
Be injected a small
amount of the dye 2,6 dichlorophenolindophenol into the
foreprai of his subjecto, measured ite speed of decolorizstion, and concluded that that epeed wse proportional
to the emount of ascorbic pcid in the blood.
Othere
since then have failed to find such a correlation (So)
end advanced the theory thet there may be other reducing
eubstancea in the skin, which would interfere with the
test*
13
In 1932 Tlllmens announced & relptlonahip between
the eecorbic acid content ot food and ltd ebillty to
reduce the dye 2,6 dichlorophenollndophenol. Lster,
thie principle vm applied to the measurement of cscorbic
odd in urine. Since then, aoms progress hae been nede
on the determination of mlntniua end optimum requlremento
for verioue nge groups*
It h&s hem shown by Harris and ©there that tsio
tt^iae^f mm®ttm of Adootfbio '&@i^ id roughly ppopo**
tlotis.1 to, the-iatake &t oertalm X©tr©Xs (4f^6y$$«74)«
Johnson n.ad 'iilva (33) found that the excpotiioa dopeadB
i©th,afot''th© ia6§iiiti^e ofth^ iatstse maM upom the
degpoe of sfcturattott of the subjeet^ a aach amall©?-,.,
l>9&Qomt®§e of a siftgle laege dose teelag ejco^etsd if the
9et»V99t^ of the gtibj©at hatre beeat pr^tirioutlijr de^lete^i*'
< For e, titao aa attempt uac m4e to aa^oao nutyitional
9tattt& hf a©t©Kiiaiag the iirias»y ©s©^>tlOBt of ascorbic
mid {45»11?)* the tevet of eso^atioJa «ow?e$pmtfias to
the ^louier' Mialtof ttonaal" hat heeh ©et at appj?03S.iaKitel7
13 to.^O- ag by verio^t vtiv&epe (43#iiSf3tj# Sdi»l9»
Bey,, ^h^ VafNl, aad Ahasi hsire d^tia^tod that th© ^©ruge
dfiity ex^rettoh fofc 'horiaal a^hledtt lit ^'bout 30 gig
('K>,3). Hfi^ie ©ni Abbfvay (^3) fouM tha'i a*ii intake of
25 Big of aacorbic sold would reeult In ail oxcrofeion of
13 wg-*
oa© suhleot of «roh»i©a ®sd ztlvft (M) «a*»#»od
i9
80 to IS© ag ©f aieoovblA acid «k©B h© tfeggi&tfijr tiaelutod
is..hie dfitl^r «li©'t fcti© ©r^ages b©tid©8 g^een ^©getaW©©*
• It -fee* toe©» #&©«'© that there is a tti*©mi tfep@'sidiaw
v&ioe o^.&'definite «s®6e'©otrfefet©n la tii® btooi fet wMcia
feseoFM© ■ &$!& .begieo t©' spije^x? ia the w^ia© ■ ia iftrge
siaoitte'(Si>B.,51*l4.}* fhlfi iwaat' thseiChoW tm*!©© fe*1 •
Alffereftt iafilvtaisle, .bttt th© average a^p©©j?e te he
about &.# to i.4 ffig pev.idO al of bloei ptiift&u Mm&
the tii©e^©e' hoe^aie "eatiwpated* ?lt&
the intfilBe && Mu£6eiit|r stoip®^# the
^api<ilir> ggnetl? hy the eettewd' tef,
XetteV-ett&e&t?? f®iM ia ^he Mh|Q©t
'a&ee^h&e aelfi «i€
©sereti©^ d^ep^ ve»y
te the See festiisg
oa s f^otly ^kosea
dliet (55)*
- Jdui&en .acid 2ilva (55) gav© vitanta 0 4efieleat
dtthjeetft gvedoally i&e&eA&lag Aeoeo of atieerhl^. aei4
amS foiaiUI he ©igaifi<&©at rifi© in ®*6&etie& .until the
Deventh day* ®3s® «©©aat ese^teted ^PMmeiilf roe© until
the «e$etofcee%th day» ^h©a a eornGtamt l©^©! wo ffe&eheAt
this pelet- they ee&ftKtered t© b© the level' at ohieh the
tif0^©0vi!i» esttti^ted *lthi 1$B® vltsals*
H©i©©ffiaiiaif tjfaa W0^0eh> ©ad t&a B©Ml©a ha^© attor^pted
t© aesoei*©' th© daily e^peoAituye '©f &e®optel# atid;.; '"(50>.$l}4> -'Jft deeerlhiftg th©iP ^etlMdv th©y fttyy- .■•.••■
If a .pvetrlettily. £attg*eted ^bleet.io^ ..■.•..s
reaatrir'ated efter hpvlng omitted all vitamin
so
C tov B certain length of time, the dose
of a&eotfbie aoi& ve^Bi^ed t© produe© sat^©*tion corresponds to the qusntity tided from
tli© body etefee during tli© irltiMiia 0 fjp©©
period* Consequentiy thla emount w^ien
divided by tii© iitttabol^ of €sjr®- %%& ©atp^^issoot
lasted, csn be conaiciered as the cieily expen"Satwatioa" is tteei* ©Kp^ftaents Is o©a§id©f,©d to be th®
condition when eonsiderable portions of & tost close
fii© yeaponto to a /rtt©st d©0©w of feooo^bie soli liaa
beon Bid©|.S!• ia8©d a» a method of a©sa:tt^ifeg a^tyitloaftl
stst© jutui yofalspemesto*
A. lid^g© doe© .(20© to iO^O 09)
is iiig©8t©& and til;© virine o©li©'0t©d to* the mxt §4
houre a&d analysed for oocorbio acid*
This method,
in it3 m^ny vsriGtions, io foimdetl wpm the principle
ttat © l8i?ge p©»o©at«.g© of tii© t©8t do9# ia ©sei?0t@d
if tfei© ti©,im©a
OCCUPC
®IP©
"^Atmrattedy* vhite almost m 9%$o
If the tissues $i»e severely depletedo
Sometimes,
several daya ©l&fa© before tb© exoivatlpn responds. to
repented teat doses (78).
Herrie and Rsy (45) found th'>t low excretion and
lack of reaponse to teat dose go parrlie1 with © history
of loir pacerbic eeld intfike ,?,nd with n state of rscorbic
acid subnutrition aa shown by cftpillery testa, but he
recommendB the teat doa© method as bein^ a better method
of measuring nutrition th«n the CBpillory resistance
method beccuee it is more truly specific and more aoeur-
21
stely qu^ntlta' r.ve end beceuse Fepeated raeGeurernonte
CBH
be made ©t aoy ttme.
Hr.rris and Abbasy (4?) found thPt the consumption
of 25 ffig of secorbie ^cld daily will yield e, response to
the test Son® generally on the first day and certainly
00 the second.
Ordinerlly, the deterraination is m&de on the urine
collected for the 24 hours following the teot dose, hut
doae hsve suggested short collections (3 to % hours)
with the argument that the peak of excretion eomos at
about the fourth hour and drops rapidly for the rest
of the tw©aty-foi**>.
Hpigkt, Wing, ldli«ttfletd« fitnd
MficLenethen (109) have found that 80 per cent of the
excretion occurs in the first five hours.
The test doses used in different studies have
varied greetly, and there is a great difference of opinion
£0 to the pereentege of excretion which indicates edequste and saturation levels of the vitamin in the tissueB.
YouEens (113) estimated that Q.n excretion of 30 mg after
i% 600 ag test dose shows that the subject is in "normal"
vitemin C nutrition.
Abbpsy, Herrla, end Hey (l) set
a 40 to 50 rag excretion of a 700 mg test dose as normal.
Fpulkner and faylor Oi) found that their "nonaal suhjeets*' (blood levels averaging i*T mg i>er 100 ml) ex^
creted SOI mg of a 1000 rag test dose in 2% hours.
Van
22
Bo&elen (10^) 4©fia©d ^seta^&ttoa'1 as the p©imt «t
v;>>lch a dose cf 250 to 40© m$ of fscorblc acid will
result fa @, ^finite rise eb©v@. 30 asg edeoretl«ii»
Wiplg&fc,
lpvi»g, Xilll^Qfle^, ana teeliQ^fttfiiea. (109) 3pep©2»i ^hftt
© tunnwil ©tfej^ot wxtt mme%® §00 lag of a i©0@ sg toufe
a©s© lm 84 hcntra ©* 400 a»g in five hours,
fov mnf
jpeQuls'eiaest Q^eii©ei» "'s&fctay&^toa'" is e©tsis$^©s*e^ the point
9% %M®h %Q p©2» ®mt of & toot €©s@ trill h@ ®mv$tQ&'
(97).
$t@rvi©k ©»^ Sa^ck oonperod this i^thoa ©f d©t©i?*
ulaiug 9o<|tt|v«80RtO' with a aetfeod in ^hl&k tbo iNibjoot
is given £00 rag of aoeorbie aoid tstilsr fo» e, p&eliaitiBry
^QQtBPotioa
B
P«MP|O4*.
^© l©w©^t roopoa^o to -a toot 4oae
after tfeio poriod is t©k©n, ©e th© mooaepo of ^estadPatloa"
fo* tb©t aubject in. oubcecmect pofeiodo vh&tk th© cwb^ect
is k©?pt on ^^Pioaa lovolo of inta&o to detemiae tl»©
nittlama toQttireno&t to pm&m® * satiation* * For tbroe
aubjooto., r©q:oiroia©nt$ &a det^rainoi bf tlto tv© mot^oio
the mm®, tAillo in t«o> their ^or© oliaflhtly feifgiQ^
ro detenninsd by tho second netbod.
WQV®
In t®3$i> Faster and AM (t0) r©po?t@d t&at fch©
amount of reduced eacorblc acid in the blood plasma
closely follows the vitamin 0 tnt^se.
Since tben, others
hsv® ooftfirood tHio oox^©i©ti©n (ii5^i7*40)»
Since the
introduction of tlie plaotia aiero tltvatloa tootmiqne of
23
Parmer ami Abt# the plssmp- love! of ascorbic ecid has
been uaed to s greet extent both In determining nutritional state end in calculatins requiremsnte.
Some hair©
tried to determine nutritional state by enslyaing the
total ascorbic ©old content of the blood plssmc after
reduction with hydrogen sulfide or carbon monoxide pnd
find no correlEtion between totial fecorbic acid of the
pi?.ana and the dietary regime.
Farmor and Abt etatod
ifis fflop© algaif to&at ■ %hm the t©v©l of t©ta"i eeteo^hio
©old ^3 a i8$$8Utti» @^ ®»tS'iti©uai etat©*
^laeaa'. l.©v©l.@ ©fid- Hid oaema^ o? feftftoiftlo «eid 9a:©s?et©d
la the urine.
^a^iiKMtui''of &$■ vlt&jBitt ft eoBit^et'Of blo^t ami v»tm oa
©f, the-- g^nt «hi«h sh^w^d a atfss&i ■$@*9QN8& fee a t®^^
d©i© (^0- t0-S@-fflg-ef ■ * 706-818 *ss* deee)* ealy-ene hed
a hleetf level of leeft than 0*65 asg pfc* 100 al# a level
tihl^b; they efcdtte eo- IhAieatlh^ no^isai mtj!ti?iti©a..' -9heF
helieveO that' the, ©nelow -level ■&&& fom© bee©- doe to at*
aansualtf i®tf i^teSee ef vifcusltt 0 fey two
p-^ocoding the te&i.
OP
tlhfee ia^e
fhef stateH th&t. eeeaAlefiallst a
bleed level may not bo IndieGtlsra ef the tatFltlomal
status: btt% iM^Iievetf tl4«.fe t*& tim fflo#i& 5*»t# tfet© iietJi©d
Wortii, ivletoMMEd^^ m& ¥@*i6it .(108) m©€© ©xl©^.siv©.
©OEipci'&tiv© stsdtee oa plcfimSj) 'ttPlEe,
AQA
BMA
spin©! fluid
€soaelL«€l®d that & ttotn&I (©♦? «8 pet 10® »^) -m& e
wry. iiwri ©se^bi© ©©14. «©at©at; of bl©©«| plfefi^a (fe®t©!»
0«4 iag pep 100 ml) are almost invariably eseocietcd with
eo^eapoaii^g ao^aal ©at eubnormsl valued foi? bofeti jBptaftl.
f ittlA. euM ©eeotbt® aeid esvpojttcm ia f tv©
IMMMW
<af *«*
& "tetMj (t®-©©'* ®ttdi ^b!&t> ttei^©f®^©'.p tii© fel©©$ pi^sisa.ia
,feii©'8© jp&sages fiai?aii8fe©8 ^a ^d[©!|S!imt© aiaS ©euuuat® ludos
of '.vlt^wia 0 .matMit©***. Xtt- tlte »S«>g© #f '0*4 feo. 0*T ng
|5©JP
t00;asi| tlbsy "i^e^onsi©'!!^. filtfe'fe -ftSLl,.asftfti te@ jstc©^..^©*1' tb©
©lial©a^ evfttoatlMi ©f e©isi»vy*
fl^owie* a»t l®^©t ,(99) f®^©^ %]fe©fe tlteVQ.^ftd..^.
^®f iBf ^5© 0©*l1P©1.©(f t®ll b©^©©!^ ,liFfll©,$>tf ^HH p'ltftglffiR .S.5t©03PbS.©
^©liS ©&, ttet®l?©.$ ©f 7S u^ ,&fid: .i©inS' bti%'is©% ® C5lgiiifi©j^ai»
©'©5?ip©l®^i©ja
©E.
I©*'©!.© febov© ^Sfesfe.
1!li©$© ba©. b©©Xi ©^ai© ^!8©0ti©s #b©©t .ubitftlt. f3?s©t'i©ii
©f fcb© bli©©d $.$ tsb© b©©% iQ©#i!uifi: f©3? i@©'fc©i'aiii5iau ms^i*
tloiial 8%ftt©*
Its life© 'b©e& ©b©wa bf. 9tttl©9 m& B©t«di!iaiia
.(18 ati.4 S4) tb®t 6f«o»bi^ 44Sd# 'wb©«i It is £i»*t .ab®©*»b©dt
©IJJI©©??-©
lo: tb© fi|ife©iaft sad; fefeeii #i©virsi- 'i>©®©fc3P6t©© %b© 3i?©4
bl©@i ©©lie.,
©JB
A fev w©i»k©i!»©. {i44f76) bfef© ai?g«i©i tbat
'©ito^©!© ■©? Hilt©!© bt©©€' t^iil. (giy© $ feot'i©?? ^i©i5^® ©f
vHHOTia 0 ttat*ttl<Hi tbsm ©itb©^ vblfce ©©1ii*pl®t©l©t layer
8^
©$> piaame. bedausd it does m©t fl^t^iat© a© t&© ottie&t
«»*■•' BsatX©i? fe^a Ol^lMaati {i8); I»©ooai8iea4 wMte <seil*
ptdtoiot tmfm fifialyeift slfee© th»y ift wqpoff&tiftit ivi^k
e»6Ka@a» ^nay&ai Uili ($%} h&v© f©.ais€ that tibe &9e$*bi«
mM t®m% ®t the wMt©' <s©il»piat©i®t lay©^ is tho lea*
ZG beGomo aoploted la deficiency GDd the fii'Gt to 2'iee
«iK>it aooMid aeii iittete- is r©:8Qffi©#* It is ©^iiemt
tb&t motfots of tMa ispj^- ^©mli sla©^ tfe© p^©$#®©e of
a lo&g ^ofttlsttdd fi^i?©^© dofieloisoy b©*t0» tkm m®%§ th®
pisSffia .lo9ol< fh® latter, twwovd^* i# © bottom ©©©.cwi?©
of ^te^ tfee diot IG# bees is tfed dsfs pteoofiiag tfe© t©8t.
Ooldesl^ m& 'ttting^P (3$) .foand that vhaa ©#©©^hie
aeid wag lugodted, tho plasma level ^^i© In @^@mt as
howp, tfe©© pevgl&tod ®l? ^oe^hed a podle at' the esstd of
tJip®© iiws m<& fell slightly ftt the e«fi of ©is hou^o.
2%®^ ^ec^iae^ed the detenaisentlon of the ttistfritlOB&l
-Qtftte. hy ttsitas. ths fe©i#it €ssi gaa^^al hehftvio^ of .the
blood owrao afto» sm vttol ^oo® of ii©^©^bie aoid.
ffi&lttfttioa. of ^othodti>; foy..g&tegijiihi^ Kiitritifml $t&%®
ilmOSi hm te@® aaii teat asd agaimot the' maiPload
toothods of dete^isiftltig vd^ai»9iBeht& ohd mtPitlooal
otftto* 1® 8e&B^«l# %&@ blood te$t8 have p^o^od; to he
tho sioet ©mtiafaete^y ©md oofivealeat* •
Stosa (.fl) ©tetee that the det8]Raittatio& of the
vit&mift 0 ooatettt Of blood pUKnaa i# the oiiqpleet
26
dependable procedure{ he also states that the meet
precise e,M dependable method of determining degree of
saturation 1$ the rate of diseppeex&nce of an Injected
dose from the blood etreara and that the method bated
on rate of excretion of a test dose is less precise bat
quite dependable.
Van Eekelen (104) believes that
the determination of fecorfcic ecid in plasms is better
than urinary studies for determining nutritional state
boccuoe it ie less compliceted end only one or two test®
are required, while the urinery test oust be determined
for a twenty-four hour output; he states thet that output
is very variable for different people*
Farmer and Abt
(29) believe the blood tests to be more informative than
the urinpry.
Goldsmith and Ellinger (35) found in their
experiments that there is e close correlation between
urinary end blood findings &* indicative of normal or
deficient viteain 0 nutrition, hat they believe that
the urine tests mf he less reliable beceuce of the high
pmount of interfering, non-Bocorbic ecid reducing sub*
stences in the urine in such conditions as diabetes or
when the subject is on e high protein diet*
Some workerc
heve found that the great variation in response to a
test dose in the same end different individuals makes
the test unreliable (**7,1©9#93)»
Some of these have
found (95,109) that instead of progressive increase0
87
in excretion with continued Mgh flosee ©f rscopbie acid,
tfeeffe is a© furteher ti$@ f«* several, iii^©^ ©!► imtil %fe®
deftes ©j?e diBContinuod for nevep&l w&&© te&H* ?fei*
tt
pl©t©att ©f ©3£@f»©fci©jR* H©iia©ffle«Ba ^©lievea uae da© t© «
f©I'm ©f ifoeifi© dyfeeiaie mttoUj la «l&i©l> %&© ©*©@8S ©f
vitfeisia dina^pefefe^ afteip *h® bQ&f
®$OVQ$
mpe filled.
Johnson and Zilva (55) ataf.ed that, tit© povcent&.ge
©xc^ted i« feigfeoy ©» lover t©©t' d©8©« fctesu ©s high ©a©e
4i3id that the sunoti&ti retrirted is higher in the aecon(5 cose
and i© a©% ladioetiv© ©f th© feru© »@«iBl*©ia0Bt»
fli©a©
ftte^tioiis 8!^©w .tli© tosewtaitity ©f ^a^eadlng ap©^ tli@;
test
S©B@
a© & tft©t|i©<& ©f (iet©i»iQiRi&g5 peqaipeiaent©,
Pipcma Ijevela
^ij©*© i© a©ia© ©©ist!r©ir©»«sr ©*©«* *&&% ©©©©%it«t©e
s
«ioi?B»lw vala©© f©2» tfe© eccorbie g,©i«| content ©f blood.
Ifeftgr •WMiHwiiowi h®v© ^©©fe ii?©¥a bgr trsrioae «©i!»k©.i?»
feft©» ©tmai©» of plsfijafe i©^©l« ie j?©l©tioti t© ii©t;ax»y
Greenberg, Rinehart, f5nd Phatek (4©) studied the
fasting pleanip levels of 55 medicBl students,.
Their
values Mag».d ^Oia d#2i t© 1*4® m$ ©©©©i»M© ©©id p©^
100 mi ©f blood; t&© fcvetfag© wa© 0*72 fflsg pet 100 ml.
Forty-?Ive per cent of the students had ple.emB levelo
below 0.72 mg.
The experiraentere concluded thet velues
below 0.72 mg were probably BUboptimel, that values
between 0.7 and 0.9 appeared edequete, and that optimal
28
values were probably above this range.
They ateted that
vrluea below 0.5 mg per 100 ml must be considered Xm*
Youmfens (113) found blood values of i.66 to '1.31
mg per 100 ml In eubjects on their usual or on e. forced
intake.
Twelve pstlento, who were suspected ©f having
e l©t* Ifttftke, ha<i values ©f 0.8S lag ^© 1*09 mg p©i? 100
lal, the average being 0.539 mg.
Van Eekelen (95) concluded from his studies that
vain©© of f&m mw t© 0*4 ®$ aaeo&Mt ®>®M pm lOo at
m® potop, £*m 0+4 t© 0*3 tag iai®d@i»at©iS 0*8 to 1*2 ag 'Vei»jr
good,, aM .t&ow 1.2 as ©'S©©ll.©3at»
l^iglit^ |iill©afl©M,. aa^l Iteeltsaatheft (109) fouM
that the plasma values cf their aubjecto i/ho repcrt.od
good to esseollstit iietas^f ^ijjt0j»l©fl
WOP©
0.S4 t© 1*5^
mg jp©i? 100 ©ov, tliftt tiioa© ,»li©'«'0p©i,-t0j| f©©r to fmijp
4lotdipy tkiatorleO' Iwd :v^l«i©t of tt^a. 0*27 to 0.68 eg*
Sou© ■©£ ttbelp. ^bj©©t$ wh© j»©i>o2»t©(ii $©©a dietaffir i&is*
feorlea had, lovela- ©f .0'.3i ettt 0*37 m$> and ttio ta»lth dlete
oon3iaere«i f&i? t© good had lev© 10 of 0.4l mg.
Slesfii 01) ©©®©id©^@d 0*5 sjg ftftttttv^ift &©id |)©3? 100
ml to fe© tli©-
1©WF
limit of noi^ml afed 0*8 to 1.00 og titet©
©vei?Bg© .seynol faetiiag vslii©^
Tfe© mm&g® mlm tp*
Me «^4©©t8 vlHi tit© iBild©st #ao®a of sow^f 1ieo-.0*36
Mg and f©i? th© severe caeofl 0.2 jag.
f®yl©x», Ofeaa©:, sad F&slka©!? (l©0) fosaad tfe© ^vefffeg©
pXaoma level of 10 ecorbutic patleate t@ h® 0.245 wg$
the range waa .11 to .55 mg poi7 100 ail.
There is some difference of opinion aiiout vhat value
shove tisauo seturation.
Kalli, Prledaan, s.n& Sherry
(8l) atste tiiet 1.0 ©g per 100 ml o? over inaioete&
eeturatioQ, vhilo Faulkner and Taylor (31} ctete that
values over lA, end Goldsmith (33), values ei'er' 1.3 ag
ludieate aacupetlon.
Wont's, M©fe®a!m.fl etUSl' ^©^10 (108) ©©©d « valnd of
0.7 ag as t&a i©¥©0t tta&t ©f aoi?aai*
igg
AOOOPMO
a0i4 psv 100' al 0©©3B®4 fe© inftitato goodt
vltdmla 0 ttiit»ltioa« aM l»9d"t&ait. 0»5* « ©1M itet<a of
dofl^ionoyv
lf@ttwoil0p ooasiteFd valtaes b@i©^ d»55 ai§ pi^ ita ml
plasma at fat^ologi^&l m$ ^a.l«es ^©low ©^^ sg ad .i0flBlt©»
ly seo^butie.
l© ^oga^da valuee of atomt 0.S ng ps? 100
at at zoofj. noTiual values i>Itliotef "^tottotlag Q^tu^atiwiii.
fyoMmitk &M Mittm®$ (35) twaolnfisfl t^ftt ^a 03?igla&l
i©v©l of 0.*? sig, Mffiebl^i 8 ^g OJ? a®2»© affe®^ & t$ot dose,
iiiows ttofjalal -^it^fflls © tgtritlom*
■ Reg©Mles«; of the vl^s 41*^g©ao© of <§pi&io$a ■ ®w<®x
vk&% eo^ttlt^t^s •eatewitlflik'* lev^lt ««• *tfe® iwoy limit
of wtomtr*.It
Q&BBB&
to'b© goac^&llf a^o^ptod tfeat pl^maa
ievsi© of 0.7 os* 0*8 tag asiMHPbt* &©i^ pei> 100 ml l&Aleate
& fi^tl'Sf©oto3i,7 tett-tx»ltlo®al ©t&t©.
50
Requirements
tn recent years, a great deal of work has been done
on the determlnetion of requirements tor the protection
of the orgenism from sourvy, for maintaining tissue
saturation and for maintaining a "satiefectory" blood
level*
In the days when acurvy was still a great problem
to mariners, it was found by medical men in the British
Navy that one ounce of lemon juice per day was sufficient
to protect a man from ecurvy.
Kellie and 2ilve (5?)
concluded from their experiments that 15 mg of ascorbic
acid would protect from scurvy and maintain good health*
Fifteen mg is approximately the amount of ascorbic acid
found In one ounce of lemsn juice.
Gfithlln found that
25 mg of pure ascorbic acid or the equivalent of that in
orange juice was just sufficient to prevett lowered
capillary resistance as shown by his positive pressure
There appears to be a great difference between
minimum requirements and values which are considered
optimal*
Pinoke and Landqulst 03) found that their four
subjects required 1*1 mg, .7 to *3 mg, 1.1 rag, and 1.0 mg
per kilo respectively to maintain a blood level of 0.8
mg per 100 ml of plasma*
51
Levcovltch ond Batcholder (65), doing excretion
studies on three subjects, concluded that the minimum
©eeopfeie fecit vetyiipwuettt of fairly ©ctive colieg© cornea
may lie at about 00 rag; with a §0 per cent margin of
aefety, the recomraended Intake would be 75 mg daily.
Helnemann (50), using the method described previously,
found that one of his subjects expended 62.6 ag per
dfiy foi* 37 day^j vhile another used 63*3 mg for 5> day$*
He set 60 mg as the aetnal requirement for the adult male.
By the seme method, van Eekolen (lO'l) fonnd hie
"daily oxpenditar©*' to h© 3§ ag efffee* h© had been oa Ms
ordinary diet and 63 mg tfhen he had a fairly large.'store
of th® vitemin*. ffer&eh (Si) W ^^ *&m laothod-j ealoulated htts daily ©^penditttre a^ heing^ 56 ^© ©fcd that. of
ao ©ight©en*'ye&r«©ld girl with so^rn^ fte 4-4 sag daily.
Ralli, Priedraan, and Sherry (81) found that in their
eubjocts, th® heat retention wa® usually maintained■on
an infc&i© of i©t> w& 'daily*.
Most of the ucr-k on requiremente had been done with
the purpoBO of determining the m$0©0'&<5ry aeonnt of
fiscorbic acid to produce tldeue 0sat«r».tion«tt A .ananary
of th© rooults of SOB© of thoee otudiea uill he founa
ih Table I.
It is not yet known whether or not it io ad^isahle
or of any value to keep the organls& in a at&to of -tiaeti©
3%
aafciirafcioSb bnt fcto©*© Is
A.
gp^at; ^eal of evltenae tfe^t
a©*© tluka. tit© lalatmim y^^ttiyeaoitfe t© protee-t fanrai se^vy
is ©©'©©st®!?^ to fe^ep tit? otfgaiiisa in %%& best of feesitti*,
'Ml^sii tB^eospJdi^diiSiS 3P©^iai3?@Bi0si't'& it i® aoeede^ysr to #ti6t©
whether those re<7,uicementc are siinimum
OJ?
«lefift©d ia tlM9' lt$kt of jMteea&t iGaotJlQig©.
optimmi re
It in- imteroitt*
iig to note ttiat tfeo recoEEaoaded fiaily allG'waBC© fox*'
9lteaUi C. 'VA9 a©t et 75 ®i& $®% th© e&uit msa ssia 7© asfoj? tlie ©dialt ^oaaa by tfte Ootamitt®©
OB
fooao And-
KiitPitioii of tfe© iatiooftt ItoiftAVOb CotfaeiX fa IStey-, i9^i*
It xiiXX b© ao^od t^at tho^© f ignsr©©
S^Q
io©at©i bet^ooa
the aftrepagfr ^epo^tef optinam ^©qiais'eae^tf and f&e ^^@p@gG
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35
Pactorc Affeeting Hequtrenottt
Study of the phyalologioeil ftmoMono of psco^blc
6«id suggests that eertein factora may affect the requirements for the vitfciiiin by asm in varioua phaeea of
health ond activity,
Hie, requireiiieut for j eeorbie aoid 1$ greatly liie^eesed
during prognency and lactation as would be expected,
el nee tlie vitemln id' stored in fche fetus end placentG.
*Biere ie evidence tlmt re^uirerueiit for aseorble acid
beoPB no relation to ege,, height, or weight in the Mult
(8i)>
ft hea been founfi thet the ascorbic acid requirement
if Falsei conaitlevably in certain febrile conditions such
as tuberculosis, j'heumetoiu apondylitis, and pneuiaonio
(82,41)*
It has been suggested that the requirement is higher
in other eases of heightened raetaboliam.
Goldemith,
Ogeerci, Bnd Gowe (36) were ©ble to maintain & plesme
level of 1.0 mg per 100 ml in 10 of 12 subject8 with
bronchial asthraa by the administration of 50 mg ascorbic
ad id daily effcer a. preliminery saturation.
Of the two
eubjecte who were not able to meintBin e high plasma
level, one was obese, and one wae taking thyroid.
Koldaev ana Gelmnn (63) found that the ascorbic
acid content of & guinea pig muscle was decre^eed after
36
the application of an Induction current whether the
sniraela had received excessive, normal, or inadequate
supplies of aecorbic acid.
The reduction was somewh&t
less in the musclee of ©niitiels which received lerge dooea
Of the vitemin than in entmsls which suffered & deficiency.
Todhunter and Robbine (.103) noticed seme evidence
from their experlraentel work that
fatigue pfter long
hours of study and labor&tory work educed € levering of
blood and urinary vitemin © content.
Sxcrotion ctudies i& exercise C3tpos*imento heve, ao
fpr, brought only conflicting report;»
S&ffter (105) fitvtAlod the ftlgMAy
Von Jezler and
'VHJQ&V
*wt*%lwk ©f
five perticipanta before ^nd after a ski race in which
© height of 2%5© metere mift &OTiB©iafit©<i'i» © ©treteh of
50 Ian.
The partiolpante Here given 30© mg Redoxon
(s^tl^etlo ^soorbie mM) for seve© ikf* before the Jpsoe
end for the six dpys following It.
For each subject the
exdretioii felt ©a the first or seoomd de^- After the e©»*
test, the average nightly excretion for the five being
166 m& 156 log per 10© sil* for the iay preesiiag
«DA
the
dey of the contest, end 115 ^nd 116 mg per 100 ml for the
two d&ye followiAg it*
This m&y be aignifleant, bat the drop mey have been
$!ae to whfet leiaem^an refer© to ■&©
n
the specific dyn»inic
37
mtton of ©seo^bie aeid,11 in vhich the peak of exeretioia
ie not w&tntp.UiQil oft,er r-epefitod high Gceec.
Eeiiiel observed & definite drop in the excretion of
his .eiihje^t© oa the fivvt tw i&ys foiloiJiag th©!^
pertielpatIon in & football game.
Fa'trndt endi ^ehussele (i?)
SB<3©
exci-etlor, studioB
©.% $e^©:«i young aem «aib|eet© who' ¥@^©' ia g©oi pfeffei^ej.
ti'ftiniDg.
In flite of the subjeeoe, the reducing power
of the %J»iEe • j?@©© m& ill t»o# it fell* affeer ®©vem!l
fowfe of violetift
©&<B$®SB®}
^.fe© elmAge vas 1© th© aame
dlveetioB f'©* eaoh ©abjaot in eaoh ease*.
Th© exp©Fi*
natttd>>d oomldl find no ©Sh©!? eacplsikft^loft^ fov tilift %h$.a
that thoi1©-!© 6 4fo«iiQtit'tttiefi6l tfjpr^^ bt le&et ie «h&%
©o«4©ei*&s th© htii3@3?ohoi^a0'Bal 9ea.6ti9fMi in effort.B
Sti©i»®
IQ
$oia9 e#id©!ie© that i& coBSiaow&hle ^aount
of ascovoiv acixi may be lost in eneftt.
Coi'nb'lost, Elcin,
aad '^se© |S.©) jpo^^t^d th©t ffrpa 0«^$ to 0.64 isg of %h©
vltonla is orxvetad with 100 ml of sweat.
EGrnstGin (8)
ffep0»t0(t tfeefc. fr^a? ©..5 to. 1*1 sag* p®y 10© si @f sweat
tfas loat by Bsatu talne laborero.
It was calculatsd on
th© haei$'. of ^©ight to** fy'dB. evaporation that thos©
laborers inlght hav© loot as much as 16 ng of ascorbic
acia in an ©ight-howJ?
$B&
of trovle*
On the Qthep heM,
Wright and Mac Lens then (110) calculatod the ascorbic odd
iMft ffom patients. In artifioial t&rew tpeatmeat to he
about Q.O'U mg per 100 ml of sweat*
Calculated on the
5i
basis of & loss through evapornfclon of 1750 to 5500 ml
in a five-hour period* the most that could be lost would
b® two to three rag of ascorbic acid in that period*
From this they concluded that vitamin G is mot lost in
&nf ©pprecieble emount through exceesive perspiration*
In the seme fever periods, the urinary excretion of the
vitamin fill to a g^oater degree than could be accounted
for by the loss in sweat.
This drop they attributed to
a probable loss through inoreased metabolism.
There is also a conflict about the effect of increased dosage of ascorbic acid on physleal performance.
Wlebel (106) noted better performance in scarcely any
of H female athletic students in a ski tournament
when large doses of sacorbic acid had been supplied*
Basu and Ray (5), recording fatigue curves of the
finger rauecle on the hand ergograph for four subjects
before and after the administration of large doses of
eecorbic ecid, reported that the onset of fatigue waa
definitely delayed for some time after the administration
of the vitamin*
Crandon, Lund, and Dill reported that their subject
could ran for only sixteen seconds before fatigue set in
when he was in © state of complete deficiency* while
after ten days* eecorbic acid therapy* the time was
increased to 66 seconds.
This may be compared to the
39
perfcrraBnce of men of the apme ft^e, which .^voragod 270
The (31.9sppeftrr.nce of blood lactate was follotred in
tb© reeo^rex'jr pe^iofl fx»©® tli® ssaa fatiguo tests > and it
was found that the lactate diseppe^red more slowly dtu'ing
the recovery period wfton tli© auhjoet wa$ in th® eoorbatie
0t©tea
^Bho inlntite*.03cfg®& conewmption per kilogram of
body weigfet* howevesp^. «&• a© hlgfeev in fclse fatiguo tosfe
fefter th®rapy tbsa it was vtiea tb© sab joet fras ia the
scorbutic state*
0r©ad©a, Lund, and Dili (2i) did »ot find ©a la*
c^essod metabolism in ascorbic ecid deficiency, as some
others have reported*
Uteere was 6 slight dro^ in aeta*
holism in the seorbntio state*
fhis dr©3p>t tfeey believed
use probably due to weight loss or infinltlon or both,
fhey reported that, in the first #oar days of vitamin
therapy, when the subject
XWB
becorains saturated with
ascorbio ©old, the basal aetabolie rate was oonsistently
lover one and a half to two hoars after injection than
It was before the injection; following the period of
saturation, the basal metabolic rate was ooadlatently
higher tiro hoars after the administratloa of asoorbio
add than it was before its injection.
With all of the conflicting reports in the literature, it is obvloas that the mechanism of the aotion of
escorblc ecld tn muacular activity and tissue reBpirption
$9 not clearly understood* but there has been enough
evidence presented to conclude that the vitamin does
have some role in these metsbolic functions.
H
■QMAfWR M
tismm m
MIS
xrasfioAmon
The question has been opened es to whether or not
human requirements for ascorbic ftcld sre effected by
muscular activity.
The Issue haa boen suggeoted by the following
factors:
1* Ascovbtc ftcld appsafs to fe&tf© e,a esee^tlsl
rsle in the general mstabolic proeesBos cf
the body*
3* Aeeo^ble aoid feppearfe t© Mve © ppoteetitf©
or regulatory action on &t lea&t two respire torr ©nuyiaes i» «^©'l,©t©i nuscle. Foptfe©^
evlcience for its importance in tla&ue respiration i* shown bf the ve%m&6& <&.&$&+
■ peerence of bloo€ lectete list the seorbatic
3» Bvl^ence feae been presented by esperlmentel
work that the requirement for atcorbic acid
IS Ffeised in conaitions aeoompsniefi by s.® .
elevated metf boliem, as in fsvoap^ obeSttf,
tliyt»j(>li therapy, ani ©xereise.
It is the purpose of ttie fclioulng ©tJS<Sf to ^ete^ain©
whether or not the hu^pn reGUirement of adults for escorbie &*id is ©ffeetei by $& leoresse ia physical aotitrity*
42
dHAPfBR SIX
Genercl Procedure of Experiment
Three subjects were carried through two experiinentel periods of nine dayo each.
Throughout the periods,
the subjects were kept on a diet of known low ascorbic
ecid content, while a aupplement of the crystalline scid
vao given.
The basic diet consisted of cheese, boef,
dried cooked prunee, end canned beets, csrrots, pears,
potatoes, and etreporated milk.
Foods taken ad libitum
«©re egge* shredded wheat, Ry-Kriap, rice, flour, butter,
nuts, sugar, coffee, tea, oeasoiaings, snd chocolate.
The ascorbic acid contents of these foods will be found
la Table II.
The diet contributed 15 tag of asoorble
©old per dasr*
In the first period of the experiment, the sotbjeets
followed their regular routlee of a f&irty aedentery
ttttddnt*8 life*
lu the seeond period, the subjeets
exercised vigoroualy for about one hour per day.
For the first t^o days of the first experimental
period, the subjects were given one pint Of orange juice,
one glass at breakfast and one glass at lunch, in addition to the constant diet.
This was to make certain that
the plasma ascorbic acid levels were brought well above
43
Ascorbic Acid Content of Foods.
Uaed In Experiment©! Diet
Food
' Weight
Canned beets
Baet juice
Cfnned Cerrots
ean^ot' juice
100 s
10
100
10
100
feasp juice
Prunea
Prune juice
SirapQfe&t$4 aailk
Cheea©
Beef
Potctoec
fotjalj
/iSGcrblc Mid
Content
3*6
.7
1.1
.2
1.2
$0
♦2
80
10
60
60
10©
100
.2
*i
.g
.0
.0
7.6
19.1
■W ^iiiint|l^i|Wil||)Bta^;y<B)irt^<Wi>>»i*Wl|Wiw>^^
Foods teken ad libitum were eggs, shredded whest,
Rjr-Krisp, rice, flour,, butter, nuts, sugei% eoffee, tee,,
seesonings, oncl chocolctio.
4*
0.8 mg per 100 ml, tile figure which Is UdUail7 taken as
the %9irel of "good nutrition. *
For the rest of the
period* en ascorbic acid supplement of §0 mg daily wes
given to AR, %M& women subject, end a aupplenent of 55 rag
wae given to KD end LR, the two men subjects.
This made
a. total intake of 65 mg for the woman and 70 rag for the
men.
Plasma ascorbic acid levels were determined daily
on each of the subjecte.
Between the two periods,
there was &a interval of four deye, in which the subjects
had a fair ©mount of vigorous exercise and la which they
kept their aecorbie acid levels reasonably high by teking
about tiro oranges per day.
In the nine-d°y period of
exercise, the ascorbic acid intake was the same as In
the first period*
Plaema content of the vitamin was
again determined daily.
In deciding on the amount of ascorbic ©eld to be
given to the subjects, it was Judged best to use the
recommended dally allowance as releaeed by the Ccmnjlttee
on Food and nutrition of the National Research Council
in May 19^1*
fhis figure was stated as 75 mg daily for
men and 70 mg for women*
A rough calculation of the
basic diet preceding the experiment showed that it contained approximately 20 mg (about half of which was to
come from canned potatoes)*
To make up the recommertled
allowance, then, supplements of 50 mg wore given to the
4§
vomen and 55 mg to the men.
litter a more careful analy-
sis of the constituents of the diet, in which the potatoee
were analyzed as prepared for serving, showed that the
basic diet contributed 15.l4 rag of ascorbic acid, 7.63
mg from the potatoes shd 7*51 froai the reat of th© diet.
Actually, then, the total daily ascorbic acid intake was
65 isg for the woman end 70 mg for the men.
These Intctcee
are above one milligram per kilogremj the intake which
Fittcke and I^ndquiat (53) found yould maintain, the plaeraa
levels of thoir s^hjeote: at 0*8 mg-iier kg*
Analysis of Pood a TTsed in Diet
The foods used in the experiment®! diet were enfclyzed
for ascorbic acid as follows:
About 25 grans of the food
were weighed, ground in a mortar with sand with a few etiMc
centimeters of three per cent metaphosphoric aeM and
oentrlfmged.
the procesa was repeated twloe and the
oaitpa^t €il^©d to 50' eo ^tth th© a@tapli©9ph©ri@ ■ ^ctd*
Oae.&alf ©f the extraet was brought'to e- pH of .'33 to
3.6 #ith a olirate huffer.
fo fonr ml of a soltttioa of
2,6 dichlorophenolindophenol (3 mg in 500 ml) In a colorimet©r tube were added 4 ml of the buffered extract.
The
tube was shaken and Inserted In the Evelyn photoelectric
eclorimeter end the, .galvanometer reading recorded "fc
15 cecoeds and at 30 seconds (readings 0si and Gs2).
The exceea dye was then completely reduced bf an added
crystal of aecorbic acid and the reading egeia recorded
46
(Gr).
A colorimeter tube conteining k ml dye solution
with 4 ml of 3' per cent metaphospho^lc acid bufferoa to
the eejne pH
GS
the extr&ct m& & cryctel of eacorblc
ecid was pleeed in the colorimeter and the gelvenoiaoter
adjusted to the reading Gr.
With tbe inatrmaent set iti
thla poaition, e, blank tube (4 ml of aye eoiution witji
4 ml of buffered | pet* cent aietftphoaphoric aeld) m&
placed in t'he colorimeter and the readiiig recorded (Ob).
fliie cor.centra^lo'ti of o.secrbie; aeld was ealetilateS aa^ordlig So 'ilie f'oilowiQg ©viustior-j
0 * K (log 0© * log ®t>)
"She Itemt&Bfc tow %hl® Wv&tfU tm%%m®mt IB 0«€Q6*
Pf'©i$©«lwe f©i»: 9»t6»ialtol*g ^i&ema A:se©3?felc A© 14 laval
8eve3?&l ii»0p# ©f ^looi (e&otifc *^ ae.)■_«*«»• eoltoeted in &
tnalt pMml. ©eRfealmliag evyttftts of llti&m osa'tets©*
ffe© .
Qlcoti was ctir^ad with 0113 turn ^f t;*® broad eaC of a
toothpick eat! the phisl stoppered and esmfS'lfiagad for
three minutes.
One-tenth of a aiillilitGr of fne clear
plasma urn plp®tt©d off ssu^a tiUritt lato a IS ml ©oal@ml ■
eeia^lfage tube, 0*1 mt of ^eoeft^ty feollei aad ooolei
m&%&t$.%lei®. m%®%> added nitli fcli© -•aae flpet^©^' JBa^ft.'^e
ppo'|©lm p3?e®ipit@,t@& vltk 0.2 ml of § per ©©&% aaetap'ho®.'*
pl»ori<3 e.oid.
The taboa vere ahakexi to iaaure -aising '
titratioii could be ceiapletGd.
The centrifuge tubes containing the daproteinized
plRsma were removed from the rofrigorator and centrifuged
for five ainutee.
Two-tenthB of a mllllliter of the
supernfitant fluid were placed In a depression of e
porcelain epot-plate and 0.2 ml of 2.5 per cent motephosphoric acid solution in a neighboring dcp;?QS8lon.
Hie dye wee run into the placma eolutlon from a microburette until there appeared a faint pint color which
lasted tow fifteen aeconda.
The metephosphoric acid
blenk in the neighboring depression wae then titrated to
a pink of aimllpr intensity.
She concentration of
eecorbic acid in the plasma was CBleulPted eccording to
the following equation:
ml dye x S x 2000, where 3 Is
the strength of dye expressed in mg of ascorbic aeid
equivalent to one ml of dye.
Standardization of Dye
The mathod of Standardising; the dye directly against
sodium thioeulfate as suggested by Menaker and Geurrcnt
(71) was adopted in preference to that In which the dye
was standardised against eecorbic aeid, and the ascorbic
aoid against iodine! the iodine being atandordised every
four weeks with a sodium thlosulfate solution, which was
standardised With the primery standard poteasium dlchromate.
The new method out out several steps in the
standardlfcatloh procedure and had the added advantage
ot ueing th© sodium thiouulfato ctanderd, which
1G
stable whoa it has oooe rccchod cqullibriuia, ratliQ? th^n
thQ leas stable loditic solution. ' Txie thi'eo following
aotliods o? stGriClGTclIsatlcn were comparod enti good checks
TOCoivee: lite mothous just deseri'oea and the metlioa of
QientiavdlztriQ dyo agaiaet an ascetic acid eolutioa
which h&a been titratou witli & sfc&nd&rd po'c&osluiu iodate
scluvion.
i'ho procedure used fcr BtasidGi'dlaiag zho dye
follows:
1G
ae
25 oo 9is tile dye solution te$e pipetted into an
Erlemaeyor t'icsk; O.f? gi^ms pota&iiam iodide cud 0.5 cc
of dilvite culfurlc ccld (1:4) uere edded end the raixtu^e
sliakon until all of the icdiiae was llborsted; .001 N.
eediun thicsulfate
WGC
?uii la from a 10 ml burette of
small bore until the yellow Iodine color had practically
diaoppeared; then 2 cc of 2 per cent starch solution
were &ddet£ end the titration continued until the sclutioii
bocrw© colorlesu.
One ec of 0.001 1 iodine ia equivGlent
to 1 til of OoOOi K scdiuci thiosulfate, end it has been '
ctown that i ml of 0.001 g iodine will recct with .
tuccrbic acid, eo 1 ml of 0.001 1 ccdiura thloeulfcte ie
egtttvalettt t©' ■0*088 mg of li«0'Oi»'bi# sola* f&efef©^©1, «b»
etr-ongth of the dye eolution ccin be CfilculGted coecrding
to the folto'&lng eQiiatiOE:
at VtoJSLQ* 9 K % 81
"'•'■ '"*'<*■!■»'■■»-. .»■«--'
25
* mg ©soorblo aei^l equivalent
to 1 ©e
@ ie the normality of the thloaulfate solution.
Stsndardizption of Sodium Thloaulfate
The sodium thiosulf&te eolation was atanclardlsod
egeinst 0.1 M potaselura dichromate about every five or
fix deye until the fresh solution had become steblllsed.
It wes found advisable to meke the thioeulfete solution
several deye before l.ta first standerdiaetlon.
The ccdlua thiooulfate atendai^dlzatlon was carried
©sat sLeeordisig to t%© ^r©®©tmr© eattltied by F^l©# m&
Ee.Ba©y in ^^©atit&ttv® Mo*>$&&&® C^enist^y*w A t»8 B!
p6l©,#&i«a' ioiSiio iS©l«itio'& (loist© tpm) tt^e p^dpajrodi
1® at' of t^ls- mae M&©<& t© a©i ai of wfcta», follow©^ by
5
Q!
©f; it ;-a. fey^^eliloti© mM*
To t&ie w^t© adddd
alotly. t?if& dos^taat c^fcwlng 05 sal «f at^nia^^ potaa^is ^aa allo^ot to: i3ta®i': out
sl^atickfomte isoliitioa.
of 6ia>e*t tti&tigut im tlM^e »iautea asa^ tBtom Allittd'd to
ml and vi^ratcci wi'ik tko sodlam thi^salfat^ ■ solution■ SSld: no^aslity ■ of i&© thiosuljrato wOlaiiosi v;aj 3altbstlid folloiiiing ©tusitioat ';?
223
dOltttlttlUl
—"'"^"ihukd;'
"
—
.,.'.■,:••.••
•■ py&, . ?©©^t|r*®iibt ©g ^f tli©"<ay& -2,6 dioMe^o^euo*
limdofij^aol i©i»©: • dissolVQi it* 9®® m% ot-httt wat^^.-^Mela
fead b®©n-^ietill0ia f^om gl^so* • 'Ihe'aolutiott waa cooled
m
a&d 20 ml of phosphate buffer, pH 6.8 added*
The solu-
tion wes then filtered and stored tn a derk bottle la the
refrigerator.
The dye vat made fresh weekly.
Metephofphor1c Acid Solution.
The sticks of solid
neid tJcv',3 rinsed several times with redistilled weter
and dried to remove the coating of orthophosphoric acid
which forme on the outside during storage.
Five grams
of the acid tier® dissolved %n a sssll amo»mt of boiled
and cooled redistilled water and made wp to 100 mX in
volume.
Thia wee filtered dad stored la the refrigerator.
The solution was made fresh about every seven days.
Sodium Thlosulfate Solution.
Approximately 24.82 g
of crystalline sodium thlosulfate were dissolved la
water and made up to one liter*
This approximately
0*1 El solution was kept in stock aad a ©.001 K solution
made from it immediately before etandardizat■on of the
dye *
Potassium Dlchromato Solution (0.1 U).
Approximately
^*9 g of pure dry fotaeflium die&romate ^er© aceuretely
weighed, dissolved la distilled ttater aai ©arefully sade
up to one liter*
Lon of Subjects
feet KD, a graduate assistant in chemistry (23
years of age) was five feet, tea inches la Height aad
weighed i40 pounds.
He appeared to be rather nervous
SI
and complained of "indigestion" before the experiment
fcogeut* Wt' v&s In dppctfpe^t good health otherwise.
Att9#
the expericent hz>& progressed, he f\nnoiincecl thet the Indlgesticn h&S discppesrea end thrt he he,d a great decl
iaore vitelity then he hed had for $090 tine. , fhls state
continued throtighoitt the entire ex^e^iaeht*
Who© -Qeee-
tiop.Gd cbout, hi© hafc!t$ of eatlag,, t% *a6 fouad thfet they
«©»•© i^teguMr m& poov* hi© -uett^i hteeltffeftt helag e,
oup of ©offee*
fht© ©nhjeet Id th©. only one of the three
fcho GjOnpl&ltteA of hel^g tlre^ iurlK^ the period of
cKereiEe*
Subject LE (26 yerre of cgc), the other rntitij felso
© gvadw&te 6o#l$t&tit ih ohoaietr^, we© § feet* i© laches
in heiiht m& weighed i%4 pounds.
1)9 nervcuo, and it
WPG
Ho, too* ^ppearea to
disclceed th&t thare wee s one thing
worrying hla &t the time of the experiments
It we5 often
very dlfflotilt to obtain blood semplea from him} thle may
heve been beceuee of his unusuf1 nervous tension or
because hla fingers were ocarred from burno.
this subject
eomplfeined of being tired on tho last flay of the ©atpori^
aent, but the oxerclee 414 not seem to tire him as it did
ewbject KL.
Snhjeet AR {19 years Of ^g©)^ the wlf© of $&+ w&s
was 5 feet, J inohee in height aaA weighed 104 pounds fit
the beginning of the experiment hut gained two end one-half
pounds during the course of the experiment.
She also
appeared to be nervous end was very active, being aitoae*
maker and student and working in a laboratory m^ of the
time.
The exercise never appeared to tire her.
53
CHAraR IV
RESULTS jm DISCUSSION
The delly plasma ascorbic acid levels for the three
subjects in the two experimental periods are ehovm in
tables 111, W90 mad V*
the average of the last four
days of each period was used as the level at which the
plasma ascorbic acid content was maintained at that
pertlcular intake.
For subject AR, the average for the last four days
of the control period was 0.72 mg ascorbic acid per 100 ml
of plasma, and the corresponding average for the exercise
period was 0.66 mg per 100 ml, the daily intake of ascorbic
acid being the same in both periods.
It iil%% be noted
that the level dropped to 0.57 mg on the last day of the
exercise period.
The average of the last four days of
the control period for subject LR was 0.60 and for the
exercise period O.58 mg per 100 ml«
The correepcnding
figures for subject KD were O.78 mg per 100 ml for the
first period and O.69 mg per 100 ml for the exercise
period.
For subject LR, there was no significant drop in
the plasma level maintained in the second period.
For
subject Alt, the average for the exercise period was
0.06 mg lover than in the first period, and for subject KD
the average for the exercise period wee 0.09 mg lover
than the average for the control period.
It 1& generally accepted that the titration method
of mee.snrlnp eecorbic ecid let plasma is prectoo within
ebout O.i tag per 106 ml.
In view of this, it cannot be
considered that the lower everagea for subjects AR and
KD for the ejcercise period are significant.
It is inter-
esting to note, though, that subject KD, whose average
level was 0.09 mg lower in the period of exercise, is the
only one of the three who complained of being fatigued
by the exercise.
It ie worthy of note, also, that the
trend of the plasma level in the exercise period was
downward in the case of each subject.
Considering the minimum "level of good nutrition"
to be 0.7 to 0.8 mg ascorbic ecld per 300 ml of plasma,
it ie apparent that & daily intake of 65 mg for the woman
and 70 mg for the men (figures which are 5 mg below the
recommendation of the National Research Council) were
sufficient to maintain this blood level for subject KD
in both periods and for subject AR in the control period
and insufficient for subject LR and AR in the exercise
period.
Subject LR required more than 70 mg in both
periods*
As mentioned before, subject LR was worried &t the
time of the experiment.
It has been noted in this labora-
tory end others that worry and nervous tension often
55
appear %o be accompenied toj a lo\i pleama ascorbic acid
level.
Thet may km® boon & footer in the case of the
apparent high requirement of LR.
It was revealed tbat SB Included very little exercise in his regular routine, while LH and AR were accustomed to a fair amount of vigorous exercise.
It can be concluded from this investigation that the
ascorbic acid requiremente for the three subjects used
w©#e jtat vaiftftft M$t&$&l<Mia&tt tef &bm% &m km* of
vig©i»oi$s ©xeyclc©; per #>?»
IB
.?ovl0vittg tli© Aitertture ©a tli© subject ©f exer-
cise in connection with vitamin C requirementE, 1% is
apparent that the experiments performed h&ve brought
conflicting .tfewltftl. Mrnml <4i) ©ad ##m Jemtet ead
Saffter (105) noted © drop la «s<i©*fei© ®0£d ©xofpetloa
aft©* ©36©ffola©.,. while Sraadt ^ad. 8oluij»«0le (16) aoted m
drop in the excretion of two and e rise la that of five
subjects.
$» poisibtilty ©f & lose of t^e witamla though
sweet in exorcise should be considered.
Calculating the
feoufflar toss wltH tfee highest flgai»es fotiad l.a th© llteMH>
tare, ©*5 to 1*1 lag, footed fef Besmstela ($)» It would
^ppeekF that the loss for the added hour of exercise for
these subjects would not exceed 2 mg, an amount which
could hardly be considered as raising the reauireiaent
sigalflcsatiy.
56
AB
mentioned previc-utly^ Koldaev and Gelmcn observed
that there was a dcereeseti r*BcoPbie p,cid content of 6
guijica pig Eiuacle eftef the eppllcetlon of an induction
current.
It ie likely that %he etiwulus uoed resuXted
in the ccatreictiou of a far' greater percentage of muscle
fibora than would be used 1% any voluntary phycicel
exercise, 00 thelz- experioient is not comparable with tills
etuciy.
It Is Quite poaeibl©, however,
that If this
€>3£P@#iradfit wo^e i-ovioatea ytUi iiibjocta wko uepo able and
Hilling '^0' ^©vot© seveittl Jkotwi a. €©7 to- vigo^otis es©***
ciao, c a@fi&it© aror* iu plaftiaa Rsco^'Die aeM yould be
notes*
It is possible, aleo, thst a greater drop mey
ti&Ve bson iioted tie.d the expo^imoiit beeri allowed to run
fop © loiigor pe^Xou of time,
SineG tha ti?eacl i'oi? the pleemG levelc of Gseorbic
GCid
ie (iQWirw&vd for each subject ±® ©;:erjeiec, it seems
filvlB&ble ',0 stigiges^ ohe.t; the intake of GGeQi?bic acid be
patdd* dtt^i^g Mrd pl^rttieal leteor ©r ©sereis©.
S7
The ascorbic field requirements for the three euhjecto
used were mot raleed significantly by about one hour of
vigorous exerciee per day, but tile trend of tike plasma
level of ©acorbic acid wae downward in the exercise
period for esch subject.
A isily IntskG ©f 65 mg ascorbic acid wa& enougVi to
maliitaiii a level of 0.7 ag ascorbic 6eid per 100 ml for
the woman subject in tit© control popioci and not quite
eiiougri to uisiiitaiii thla level in exerciae.
A daily intake of 70 mg of the vltamla was ewfflcient
to maintain this level for ono of the men subjects in
t'ae eoati'ol period and hardly eufficient fov the exorciee,
perioii.
An intake of 70 mg wa* in^nffiolent to maintain
this level in the other man subject in aither period.
58
***** in
Bally Flasma Ascorbic Acid Values
Subject AS
©ate
Aieorbl*
Add Intake
Orange
Orange
65 mg
65 mg
65 mg
65 mg
(4 100
tee*)
65 mg
i/ai
1/22
65 Big
65 lag
1/23
1/24
65 mg
Average ©f
1/15
1/16
1/17
1/18
1/19
l/SO
Juice
Jtilee
m
• 56 aig/lOO ntt
1*22
1»I0
g pot©-
•rr
.71
*67
.72
last
Orange Juice
Orange Juice
65 aig
6$ fflg
2/1
65 mg
2/2
65 mg
65 8tg
2/3
65 lag
65 mg
2/5
Average of last
fear days:
t/n
Exercise
*92
*73
torn days:
1/28
1/29
1/^0
Miasma Level
.72
1.04
.61
.64
.75
.68
.71
.68
Badminton
Bicycling
Bicycling
Bicy&ltsig
Bicycling
Bicycling
Banning 1
Bicycling
50 mln.
one hour
one hour
one &onr
one hour
one hoar
1/2 miles
one hour
.57
.66
aaffir^'j.r/r'rr/aa'jjtfjaisaaf.-
Table IV
Daily Plasma Ascorbic Acid Values
Subject LR
mtwmi+mi ttmmii mhimw*mm*+i*i**t*^mHmtm*m*
VMMfMMtM*MMHm(«4MWtoi
Aseo^ble ■*.•.
Acid Intake
Bat©
Plasma lievel
Exerciee
mm+wmmm+**mv*mm*'i**m*m****+<*&im»ii**~+*™
1/1$
1/16
1/17
1/1%
1/19
1/20
1/21
i/n
1/23
©paeg© juice
Oraag© jute©
70 mg
0 mg
■I+ 100 g pota-
70 IBg
70 jag
_„
of last
four dtye:
Orange juice
1/28
1/29
1/30 7@ mg
7® mg
2/1
2/2
70 mg
70 rag
70 mg
2/4
70 mg
a/5
Average of last
*+mm+*mi<i*+mi*m*m
• 71
-67
.68
• 57
• 53
.61
70 mg
7® fflg
f©u# days1
.48 mg/iO0 ce
»93
1.04
.60
.71
1.07
.60
.54
...56
.35
.65
.57
On© hour handball
One hour handball
One hour handball
One hour handball
Running 40 rain.
Running 40 min
On© hour handball
On© hour handball
.58
mmfm^<m'i*t'tmmuimi<ti^>^mt
i'itiii<iiMwiiiwi'w
60
Table V
Itelty Piseraa Ascorbic Ael6 Vfeltt©©
Subject KD
Bat©
Accorbie
Pc'M Infceke
Pi&mae lievel
1/15
1/16
1/17
1/18
1/19
1/20
Ore-tig© |ii©©
O^&ftg© Juice
70 ajg
70 ©g
70 rag
»ST ag/lOO ©^
.86
1.15
7© m
Bserdlee
.Si
,78
(■*' 100 gf potato©©)
,78
1/21
70 mg
7© ag
.32
1/22
7© mg
1/23
.72
1/24
♦ 78
70 18g
&vmm& of tmt
torn toys:.
.78
1*16
1/28
O}?©©g0 Jiiic©
Otetxg© Inle*
1/29
i*a5
70 rag
,72
1/30
1/51
.58
7© m
2/1
7® m
2/2
70 ajg
.72
2/3
.71
7o m
70 ia§
.68
*A
70 usg
2/5
.65
A?©s»ag@ isf last
*69
Ola© how* feunab&ll
One hour handball
0»& feou* fesudbsll
Oae boQ» teiKifeall
EOBalag 40 islu*
fiottBlog 40 mtm*
me ii©ui» ImnAtoXl
One hour hendbdl
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.TOUP* J6ffl©i»*
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V.O. Cor lib lee t, Theodore, Klein, R.I., and Pace, 3LB,
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3*1853-^, i936* @li©ai» Abatr* 3'0t7649, 1936-. .
SI. Cvftftdotty ^.'H** Lund, CC, end Dill, 0.B* BlEpsifflr; oentat fe^ien tourvy.. Ham Magi* Jour. Had* 23*353*
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€.ir.* aa4 AM*. ii.f* l3©fcei?iaiR&tioa of
i?#^U'eei aftoospbift mi& in sffla1ii.am©M©ta of feiooi*
tew« Soe. Bxp* Biol* Hed* 34»i46-i50r 1936.
^)MAMV»
31. Faulkner, «J',M., and Taylor,, F.H.L. O'jBo^vaeions on
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'
33* Fittek©, M.t.t tm& tfttMtniiltrj V'.'I*.. fU© <lelly iatake
of aseo^Mo acid required to maintain afeoqueie m&
optimal levels of this vitamin in blood plsarcs,.
^«io>» shttrltioik ^i48>490» 19*2*
3^* Flori, k*®+ Aatitoxid aotlon of sio©i»bie aeid aad
of.extract of adrenal cortex against diphtheria.
Rev* tiiA* Fediat. 37«S77, 1939* Bat*itio& Abdtv«et6
^nd ievs* 111.236* l9*t*
35* §©ld$Biit&, 0*A.-|, ©mi Bllift$9*> 6*F* Aseo^bie ©old
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teot doce of vitamin 6* Arch. Interr.ol Med. 638
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36. Goldsmith, G.A., Ogaard, A.Q.. and Gowe, D.P.
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67:590*§96, 19*1*
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40. Greenberg, L.D., Rinehart, J.P., and Phatek, N.M.
Studies oh reduced ascorbic acid content of the blood
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1936,
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44. Harris, L.J., Mapaon, L.W., and Wang, Y.L. A simple
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45. Harris, L.J*, snd Ray, S.N. Diagnosis of vitamin C
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47. fart Bell, tf**r*M. # and $rvia, J.|»* Flasoa vltamla 0
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Amer. Med. Assae. 116?669*674, 1941*
vltealn C metabolieia of four pr'e*tJChool children.
*rotir. Bi&tHtioa dit$09*514• 19^1.
3aiO' ©iteiteticm ©f Vit6mla,e .is aoraai individual'
f ©lloniag.' '0 Mtt|Mi«96^t# -Qtia&tltetitfe aaiDlaiets»6ti©a
tA the f.©ria''of ©Mag© $kittot .itw&Zml®. aoid by
mouth, tad oovltemic sold Intravenously. Jour.
■HttWmpft 11*135*1*S #.1936-*
50. Holneunna, Martin. Hum^a requlremonto tm vlt&nln
0* Btoefcora. Joa?* 30ja299»S506> 1936*
51. Heinemann, Martin. Requirements for vitamin 0 la
iaaa* -Jefi^« Clla.. Ia^©8t... I7i 671-676 f 1933*
32. H0ia#3&aaa, MUrtla, 4ad H&aid*. F*^» Fa©t©i»# tftftt
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to cells la human blood. Jour. Clin. Invest.
191*69**73* lp*0*
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54.
Philadelphia,
A.F. * aad ©©a^amia;, H.S* tJ^iaa*? ©x63?©tioa ©f
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IQ-S©,,
55. Jclmaon, S.W., and Zilve*, S.S. The urinary excretion
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Biochoiai* Jmv. 38; 1393*1*08 # if3**
56. ifellie* A«V*» aad Sllta^ S«9» SSi©' 4©ti©a ©f !•
a*06Pbia< a^id ©a tie ia yitye i»es|>ii?atl©a ©f liv©^
tissue from guinea pigs on a restricted diet. Bio*
stei. Joaf* 35 < 783*765, 19^1, •
57. Xellie, A.E., and Zilva, S.S. The vitamin 0 requirements ©f aaa* Bieeheau Jow* 33iiS3-l6*, 1939*
58.. Slag* ©*0.
vitdmlft €'■.
19*1.
duMleai :a©t|i©d» f©* ■ d©;%©rmiaati©a of
lad*- lag# ■eheai.; Aaal« .M» 13id$5*627#
•
59.* Blftg*. ©»0« ffe©. ^©aiet^f of vit&nia 0* fha Vitamiae.
Gfel©,&g«?i, Aawrtasgi SadKial lA8s©©latl©aj 1939*.
60* King, C.G. The physiology of vitamin C. The Vita*
mina. Chicago, American Medical AssociatioQ, 1939*
61,. Ki&e* f ♦#* ■ ?it©iaia CL es:$©^fei©' fieid*
E©#'©* lot838*868* 19$6tf
-68* Kiiag. €*©*v nad il*&t6&* l$»lt«
fhf«jl©l»
^@ iftfXtt©^©© ©f HtQ*
6?. ftoMaev* BM*, ■&&&$&.%&&&■? 1*11* f!a© effeftt of
etreauoua work oti the ftscorbic ecid content and the
ei-.Idf'.ti.on-re^ucticK procesnee In the muaoles at
various ^esreos of vltamla 0 scturatioa of the>
■ ©^g^mim* maia* $imk<m* ■mm>- iit^iis 1959*
XHttv&tKHR Alwtws^i* mi 8ew» -9^37.» 1919*
A €©fe©t tn ita© ia©t^te©li&3aa ©f oapenatle ^eids la.
pttematove iaf0»tar flh© IP©!© ©f vltanla 0* Selea©©
9Ot680*681^ 1939*'
6$. I»©v©<wl^, SteftiAtta^ <mfi BetQlieM©^, 8*ti« A&©©j»bl©
mt<i Gmmtim. &t 'taMWtt l©v©t«j ©f istMlE© ^s yei&ted
to capillary roEistance, dietary estimates, ©nd human
^©tul^eaGftfe©* *r©tar* W«fc*lti©ft. t^*399*^0Tp 19^2,'
66, Loanos?, E.L. Studios ©m heaoglobia regeneration in
^etiealt wit^ vitcaoitt 0 dafieie&ftsr* ^©u^* 01in»
li3V©«tj* 19JTB7» i9*0*
oeitipv^*
^©ur.
ABI©»*
Met* .Aadoe*
11OI66>»6O8«
i9^i«
68* fittMrsliji
f&aaik* AmtOfWM 8*14 && a ^r©©ai?s©F of 99191m
©©a^;l©ai©ut* Batar© i3TJ-6l8-6i9.j 1936*
69. I3@t©v©^8^ f«, Oanaoft* f.F*-> &a$ Wlgfeit.,. 2»8* Wtsmla
0 $©fi©i©®©y**©J!fcl©al ami ttioi^^peutil© pvabloas*
»@r* 3*m* :W©d* sal* .19Tl34©*?31* 1939*
?4* W&M&mf, l*ir#> aad .SfeeppaM, «* $&f0l©t©gl©al
^vopovttaa ©f asaavbla ■ »al4» j©u^.- Suttiti©® IT*
99oa« M* 1939*
71* Hfdtta?ia»» LI*,. ttibA-(hn9*a&%* I*i* SiMMte^dHtfttlaa
©f 2>6-DichlorophenclindopJienol--an imppoveci method*
Ind. Bag. Ghem., Anal* Ed. 10:25-26, 1938.
72* 'StiMli®, l*.Si*,, a84 Sttt3<a»-f A^» $he d©t©s?ffllaatl©B
of ascorbic ecId in pleeme. Jour. Blot*
lt»t:673'-606, 1937;
73. Mirsky, Arthur, Swadeah, S., end Soskin, S. Total
ascorbic eoid content of htunan blood. Proc. Soc.
mp. MUX* m&* 32^150-1151* l$li.
74. Mitchell, B.S.« MerriEm, A., end Batchelder, E.Ii.
The vitamin C etetua of college women as determined
by urinary excretion. Jour. Home Soon. 30:645-650,
ms*
75. Ifetrdkeal* 0. Influence of ascorbic acid on liver
function and rautuel relation between vitcmina B^ and
G* $&+ Jo«l?* Gaetro-enterol. lljl»6, 7*1S| 15-23,
24-30, 31-39, 40-48. Nutrition Abstracts and Revs.
±04 $28/ Wl.
76. Neuweiler, W. Remarks on the question of the vitamin
0 requirement. Kiln Wochenschr. 18, 7&9*772t 1937*
Nutrition Abstracts and Revs. 9:446, 1939.
77* Woggle, O.R., and Wynd, P.L. A possible role of
vitamin C in nitrogen metabolism. Amer. Jour. Botany
2Til7 Siipple., 1940*
78. O'Hera, Patricia, and Hauck, M. Storage of vitamin
0 by normal adults following a period of loir intake.
Jour. Utttrlfclon 128413»427» 1936.
79* PiJoan, M., and Klemperer, P. Determination of
blood ascorbic acid. Jour. Oils* Invest. l6:443*
80. Poncher, H.G., and Stubenrauch, C.H. Intradermal
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Aosoc. lllj30S*3O4i 1938*
81. Re111, Elaine, Friedman, F.J., and Sherry, S. The
vitamin C requirement of man* jour* Clin. Investigation lai705*714, 1939*
82. Rinehart, J,P. Vitemln C nutrition and metabolism
in rheumatoid spondylitis. Jour. Clin. Investigation 108470 CFroc.) 1939*
83* Robertson, W.vB., Ropes, M.W., and Bauer, Walter*
The degradation of mucins tad polysaccharldes by
ascorbic acid and hydrogen peroxide* Biochem. Jour.
351903-908, 1941.
84. Robertson, W.vB*, Ropes, M.W., and Bauer, Walter*
Enzymatic hydrolysis of mucins by a bacterial enayme
and by vitamin 0. Amer. Jour. Phyeiol. .126:609,
1939*
8|* fteff| f.S** ©ad Gleeebpook, A,#* The therapeutic
application of vltemln C In perldental dlsosee.
Jour. Boy* Uavftt Mod* Sesv* a5t3A6'»3ML 1939.
Nutrition Abstrpots and Reva. 9:1045, 19^0.
36. Rosenberg, H.R. Chemletry and phyolology of the
vitamins. New York, Interscience Publishers, Inc.,
19*8.
37* Rotv'-cr, H. Co termination of vltemln C lit the living
©^ganiea. Hat^e i3$*7i7* 1937«
88. See look, R.R., fetM&$0)&j J, Jr., ftnd Bsslnskl, D.Il.
Further analysis of the role of aecorbic aeld In
phenylalenlne and tyroelne met^bollem. Jour. Biol.
mm* i40ti3>l6©* 19*1*
89» Mfe'look.^ M.M., s^d SllMiPSteim,- BGMfts^* $1® ooati?©!
cf expsriaerjtal c.leapto^i^rie, by msaris of vltemiR C.
Science 90:517* 19J59*
90* 89AlMls» E'#S*, aad Slib©j?©t©i8a, S*B* ■ fto ©xoretioo
of hoffiogeatielo sold ar^ ot^ar tyroslti© metabolitee
by the vitfiiila C - deficient gunlnoti pig. J©ar.
Slol. Chem. i551451*^58^ 1940*
91. fife&v* i*B.,, «s^ fljelafedo*,' 1.1:* .ImpoftSBe© of th®
e©i3tsltl2ati©B jnaetefelcm. 1®. th© cllaic©! pfe©Eioinemon
of allergy, toer. Jour. Diaeasee Children 58:
581-585, 1939.
93. fltoespasg*- K&rgarot, ami :sS©HeJ»s?f, ^*¥* ^©' fliyalolo*
gicel properties of ascorbic ecld. Effects upon
water balance end upon body composition of guinea
■ plgt« iioofee®* Jour. 33*6§§-6'|7j. 1939-*
93. Slgal, A., and King, C.G. The influence of vitamin
0 deficiency upon the resistance of £ulne6 pigs to
fitpMfeevla toxtB* ifw*. fimmm©!* l^pef* fh©i?a»
6itl«9-j 1937.
.
9^. Sloan, A* A comparison of methods for detecting and
^reding oubclinicsl ocurvy, 2OUT. Lpb. Clin Med.
$5tiei£*l686j 193S.*
95. Smith, S.L. Human requlromejits of vltamic, G. The
Vitemifie. Chicago, rv^i'iao.n Medical Aecociftlon,
1939.
96* Snov, G.A., eud Zllva, S.S. The actiosi of l-f.acorblc
aeld-oii tbe la' yttyer - yefpjLgfefeloa of liver tiesue
fs»©m galnea, f5fa-©ii 6 j?©Bti»l©t©d diet* Bloafeeau
iom*- M»787*8@0v .19*1.
97* StdpiwiWj, D:^,, ^Bb& i&wl©^, 1..1.. ftid1 pfefttttoa of
roduood ascorbic r.cld In biood. Jou* . Blol C^m.
11SJI653*6$8, 1^36..
9@* Ufeetiapt, ■ip.P*,- ami'lieSfia0»tli| J.S. ImtFev^aottda-eeoylsie 461d .!» eaiipe&ittettfttil ae-ttt© ^emo3?rlifeg©9.
I^noelt slOtSiS-SgO, 19^1*
99* Sto^vi^fep- 0*^*- emd HswiGk, a^.H* Bff^ot of conti-olled
AJMoi&ift <&«W iigeiDtioa uforn wiiwwy exe.rotloti and
plasma conceptaction of ascorbic ecid in normal
10®. fteylWj F^H..!**,, ^fea-805, t9S»2»©tt«e> 6ad fottllt!tt9*,,^*Bl»
Estijnatloti of reduced ascorbic &cld In blood serum
and pleema. Blochem. Jour. 50:ili9-ll?5, 1936*
101* 9&ffal» Sbuc,. MWI Sa^vofp 3*0* SffdOt of ab»©l»!;@
alia fart 1^1 ?ii6ni& © <30fiol©ia©3r ©m ij®'®l.i8g. of
^©tt-ade. fro©.. Bo®* Sacp* Bl^l* »©<!. 38i5l8»$25.
1933*
102 ^ f©^miLta*,;, l.U* ffe© uetr©:s» KnovSddgo of vit&mlR. G.
la Hoaltji end dlnsoato. -iToiil?. itei©.i»;* Mofcat* MeoO'*
16 Jl**!!, 19%0.
iOj* fo#seBfco*,., S*®*.-, ae^ Botefelu** Ruffe 0* ®l>e©!»vmtt0n®
on fclie emotmt of aeco^bic acid requipesl to mr>int;ain
tisaue soturatlon tm ikMBttftl ®.«lal:ts. Je«9P« Btotvitlott
19tSo>»t70j, 1919* ■
104.
i©kol©»^ iifeiPi©* 0'ii tlio. anottttfe of eeeo^blo aol^
in 'blooa. ©»i vuMnift* jh*. daily itows» peQttlvemeiitd
foj?' a«e®j!»feie sola*. . ll6olt©-®« #®aip.- 3@'t@g9l-22989
1936*
lOfU ^011 tjreaie^, A*,*. andl-Bftfftoip^ <?* Vli^ffitix t—Bddei'f
:b&l dlttatAllge* Sf©i»t,li0:h©r .JteiMH^Ieri^tdiig. freltteer
mi, WoolionBeiiF. S9*'332*333, 1939*106* Wi©^©l, S* fat5©»0ii^i«»ng©^ ib©r Vitfesln CJ^^BeiaeittHgea
feel SftMrtattttenfeltHMUNu Ii0ftt» H&Afr lf««lum0dkr« 6S8
6<l, 1939'*
TMI
107. Wclbech, SB. The pathologictl chTr.ges resultiBg
from viten>in C a.aficlency. Jour. Amer. Med. Assoc.
108$7*15, 1$37*
108. Wortis, Hermpn, Llebeman, J., and Wortis, Ethel.
Vitsaalii 0 In the -bidoii. spinal fluM,, m& ttvlfie«
^««r.. Aa^r* Sf©^* Asawu li^*l@96-i89t, 193S*
. Wright, I.S., Lilienfield, A., and MacLenathen,
taMk* tn^tmt Hei*' 60<264*27t» 1957.
110. Wright, 1,0., c,r>c? Me c Lena then,, EliEPbeth. Excretion ®£ vitamin' # 16 'fciio awafe* Jow*- tftb* 0lte/
fteft* g%8S©^*i©S.J> lf3f♦
111- Tft^oMlESr'* l#»tlfe, ASaMute^ ,P>, «ft6 Klmg, 0*Q# file
vitrmln 0 consent of hurapin t58aues. Jour Mol.
112 ♦ ^ItoyfilB^,, S» ©s tftj» l&ftiaea<5e of vitemin C ©a
AAft&liylaetle shock. Eltfea&ftt©' A^oh* txp* l©a»» ift
17-37* 19*6. StetPltlmfc Afeet^sot© e,m S©vg# If :96fl
I9%d*
115. Youaiana, J.B. The present etatua of vitamin defic?.encio8 in nrftctlce. Jour, Amer. Med. Aeeoc. 1081
i5*S0, 195T.