THE ASCORBIC ACID OOMTBHT OP THE
BLOOD SEHUI OP ADOLESCMT SUB^SC^S
by
A THESIS
submitted to
OHEGOK STATE COLLEGE
in partial fulfillment of
the requirements for the
degree of
MSTEH OF SCIEHCB
Juno 1949
&F?mmDt
Professor of Foods and ftatrition
In Charge of Major
lead of TOpsFtaent of Foods and Mutrltioa
Ghainaan of School Graduate Oonnaitte©
IT
Sehool
>Ni^rriiuiWTiiiiJii>irifii«MUiiiWJTirBrfT>»ftni»it«i ^jan^iiTrtrir ifli
Dean of Graduate
Inn uunn-'U i
ACKNOWLEDGMENTS
The author wishes to express her appreciation to
Dr. Clara A. Stortrick, Professor of Foods and Nutrition,
for her direction, encouragement and generous help with
this research.
She <also expresses her appreciation to Dr. Margaret
L. Pincke, Head of Foods and Nutrition Department, for
her help during this study and for her critical reading
of the manuscript.
Cooperators in this study were Bessie L. Bavey who
analyzed the plasma for reduced ascorbic acid and
Ruth Coffey who was responsible for the analysis of food
for reduced ascorbic acid.
Their interest and assistance
in this study are gratefully acknowledged.
H&BIM OF
oommss
Chapter X IfTHOWriOB
Historical Background
*
fh® Distribution of Aseorblo Aeid in Pood..
*
eheuBlcal Hatur© and Prop©rti@s of ikseorblc Acid..
Physiloldg^ ©f Aseorbic Acid*
Stetho&s of Ascorbic Acid Aa-say
Biological Methods.........o..................
Chemical Methods
Methods of Assessing, th© Level of Ascorbic
Acid Ihitrition.
12
Chapter II
PURPOSE ©F TEIS SkVE&HIGATIOI...........
1©
Chapter' III SXPEfiXMEHm ■
Description of Subjects •........., .'*
flan of Experiment...............................
Control of Ascorbic Acid Eatake..♦.»...**........
Befceradaation of Asoorbic Acid ift Blood'Serum...,
Equipment
.«
Reagents......................................
18
19
20
21
21
22
Procedure
,..
Table IA. The Optical Pensitiea of
Standard Ascorbic Acid Solutions
and their Mean Deviations........ c.
Chart IA, Standard Curve for Ascorbic
Acid
.....o...
..♦
Table IB* fh© Optical Densities of
Standard Ascorbic Acid Solutions
and their Mean Deviations,..*.
Chart IB, Standard Curve for Ascorbic
Acid.
Table IC, The Optical Densities of
Standard Ascorbic Acid Solutions
and their Mean Deviations..*.
Chart IC. Standard Curve for Ascorbic
Acid
Discussion of the Hethod
*.
Chart II, Ascorbic Acid Absorption
Curves (with three concentrations),
Chapter jv RESULTS MB DISCUSSION
Table II. The Mean Ascorbic Add Content
of the Serum and the Mean Intake of
Ascorbic Acid for Four Adolescent
Boys on Three Levels of Ascorbic
Acid Intake
I
3 4 ©
9
10
24
:2©
30
31
32
33
34
35
37
38
42
Table of Contents - 2
Page
fable III. Dally Serum Total Ascorbic
Acid and Plasiaa Reduced Ascorbic
Acid Values„ Their Differences
and fcfo© Daily Mean Values for All
Subjects
,.*«....•
Chart III. Daily Serum Ascorbic Acid
Values of Pour 18-year ©Id Boys ©n
Known Levels of Ascorbic Acid
Intake
.....<.....<,.........
Chart IV A* Daily Serum and Plasma
Ascorbic Acid Values of T.C. on
Known Le^ele of Ascorbic Acid
Intake............ c
Chart IV B. Saily Serum and Flaaraa
Ascorbic Acid Vsluea of ©.1. on
Khotm Levels of Ascorbic Acid
Intake.............................
Chart IV C« Bsily Serum and Plasaa Ascorbic Acid Values of W.f. on ■
Known Levels of Ascorbic Add
Int®ke..*••,................ ..,....<,..
Chart IV D. Bally Serum and Plasma
Ascorbic Acid Value® of S>»R. on
KnoRjn Levels of Ascorbic Acid
Intake.
*...........
Chart IV B. Mean Serum and P&fta&a
Ascorbic Acid Values of Four Boys
on Ihom Levels of Ascorbic Acid
Intake..............................
Chapter
V
IHTBRPHBTAIIOH QP STATISTICAL MALYSIS
OF THB DATA.
..
45
43
46
47
48
49
©0
51
Table IV. Statistical Analysis of the
Data to Determine the Sl^aificanee
of the Differences in the Serum
Total Ascorbic Acid Values of Three
Ixperimental Periods................. SS
Table
Chapter VI
V. Statistical Analysis of the Data
to Determin© the Significance of
the Differences betT/een Serua (total)
and Plasma (reduced) Ascorbic
Acid.
55
SmffilAffl MB CORGLUSIOHS.
BIBLIOGBAPIY.
5?
59
fM kSQomio mw ammm op
THE BLOOD SBSOT
OP ADOLESCEKT SUBJECTS
omffm i
mmwoftm
Klatorlcal Background
for many years the cause of scurvy, which so often
was observed among explorers, crusaders, soldiers and
sailors whose food supplies were limited, wad not known.
In 1555, according to Hess, Glaus Magnus published his
^History of the Northern Hatioa** and deaorlbed the disease
which prevailed among the soldiers in the camps and the
prisons.
Special treatises on this disease were written at
the time of M one sens by Eohtlus and Wierus.
They also
recoaanended many dietary measures which we recognize today
as most efficacious.
It has been known for over three
hundred years that fresh vegetablos would provide a potent
remedy f«>r scurvy (less, 'SO).
In the 16th century, the sailors of a Dutch sailing
vessel were attacked by scurvy,
fhen they consumed lemons
and oranges an apparently miraculous oure resulted*
In
1600 Lancaster and his crew included lemon juice In their
food supplies to prevent scurvy (Hess, tgO).
During the period from the 17th to the 19th century
Glisson, 1633; Ingerslev, 1871; and Cheadle, 1870;
described what they believed to be a disease which
*
aometimos was not differentiated from rickets.
In 1883,
Barlow reported m finalyeis of 31 oases of what it© boiiav©*!
to la© infantile seurvy {H@e3f *20)*
In 1841, BuM tistinctiy stated that tla© antiaoorbutic
property posseased by oertaln foods tmst be a definite
aubstanee xfaliih olgfet soon b© diseo^ored by orgiwl© cv
.pfeysiologieai espdrlbmito (S^eraan* H®)*
Mn$ fwlfilled
i«dd«g pspediotion by isolate and Mentifyiag.vitttain #
la %®m*
loint md Fs?@lio& {3L907) '#b$.©wfd a ais#:gis© ^alogomss
fe# seur^^ in aan from a etudy on guinea pigs tsfeioh -word fed
It. 4i©t devoid of gre«a$feaf£#« fen ytars later th© matiar©
of a *oa*entrttt& ©f as aEititoorbati© aiabstino© &tm X^moni
v&n •0t'sdi<ad by Sard^ri Mid sil^a Clfisj..
from 10S0 to IMS th« ?aet^#€ for m«» isolation of
praetieally par© asoorMo aoid f^@B oabb©,^ wat desoribi&d
•by ^-gopian {S©s<tob©ri, *48)*
.|sa IfSt Sh©r«ia d©f©lopad a prevemtiv© bi#astay
a#%hod tasfcag gui&ea pigs aa ta&e test sninel*
By his jMt&od
h© was abi© ts dettrmin© tfe© vitamin C iietsney of different
foods*
In 1929, 2il7a foimd that t&<&r# ms a elos© relation^
ahi# l»ottNH» t&e rad»«ins j^owsr of the vitamlm -and i^a
aatiseorbutle pot©ney#
fh© ©teMical naturoj, isolation and
identification of irltamia 8 w©r© reported by Waa^a sad King
5
in XB&2g and qslokiy eonflnodd hj St^nt^yoyg^i.
fhia was
soon followed by tlMr ayntlieala of vltaain 0 by lololist^la
and Sasrortli la 1998 (SosdnbAr^
f
4i) an<l naae^ aaoorblc
acl<l by Saent-Syorgyi .and Hawoytli <i933)*
fke gtatrlbtttion o£ Aaooi^bie Acid In Foo<l
MtihdQ^t asoorblc aoia la widely <Sistrlb«tedi Jn food,
plant tissues contain nuch more of this vitamin than animal
tissues.
Somo of the beat sources of vitamin C in food are
oranges* lemons, grapefraifc* taagerinea, fresh atrawberriea,
green peppers^ tomatoes, raw oabbage, splnaoh, Srusaela
sprouts, kale, broccoli, cauliflower, dandelion, sorrel
leaves, endive, and bead lettuce,
from Btudles on guinea
pigs, it haa been shown that fresh vegetables differ in
their content of ascorbic acid.
In general roots and tubers
are comparatively inferior to tbe leafy vegetables in anti-*
aoorbutio potency (Sarden and 211va, *18j Sees, *S0f Beaaey
and King, 133 and Beaaey, '38b).
Freahneaa, age, type of aoil| season, and matarity and
variety ell influence Mne aacwblc acid ccaatent of vegetablea*
Certain varletlea of tomatoee* for example^ were
groran In t&© saiete soil and under the same envircraiaentai
conditiona but some contained twice as much ascorbic acid
as ©tber varieties*
$3&ia is true for most vegetables and
fruits (Beaaey, *$8bK
From tbe results of feeding
experiments ©a guinea plgt* folly ripened teaetoe® showed
greater aatiseorfett.tlo pateaay 1SSA& tiiose ^i^fe ^©re slightly
grQ^a {Hes9.# *80)*
Bfoat cereal graina asad Xegn&es are poor
ao^rcea of viteaala © tii©6 they are la. dry state but possets
somt ^antlseortrntifc potency wtoa sprouted (l®s£ie.y^ *3Sb)»
■Milfe Is low Isa aseorble aeld edntent*- Curroa^ Barlow
ejad 3till realized that milk was iastaffieiexit to proteet
infants ageimst sourly.
Better, eggs and cheese eontain
no ascorbic sold (Beaaey, '38b}.
Cheaical Mature and I'ro^ortiea of AaeorMe Aeid
Aacorbic acid is an orgatiic Gcict ;v>iich ha a a slightly
sour taste in solution and s'liows acidic properties*
Crystalline ascorbic aeid, a white, odorless substance whidi
melts at ItO * 19^0» is quite soluble in •water (one gram
dissoives in -3 oe. of water), less soluble in aloohol (csae
gram in SO ec* of absolute .©leohol} and insoluble in
bentene* ether, chlorororm, and similar fat solvents*' 4
typical ultrwiolet abaorptiom speotr\M sdtn a auKjdmttn ©f
.063 iMi and a small band between 350 and 400 mi ia ahom. by
vitaiain 0 (Bosenberg, *4.5)«
®ie ©zaplrieal formula, of aaeorble aoid I,s OglgO^.
$eing a monobasie acid, it gives the well-defined salts of
type C@i70gM, Hhe ^oxidation-reduotlon potential of
ascorbic acid is such that it is reversibly oxidiaed to
dehydroaseorbie aeid'8 (SheKBan, '46. p. 327).
The follow-
ing formulas represent l*aseorbi© acid and dehydrosscorbie
acid?
0=0 —,
0=0—,
I
HO-G
II
HO-G
I
H-C—
I
HO-C-H
o=c
0
^SH
0=0
+2H
H-C—
HO-C-H
CEgOH
ClgOE
l*ascorbie acid
dehydroaseorbie acid
fSa© eharaeteriatle property of ascorbic acid ia its
strcmg reducing property.
In th© crystalline form,.' it ia
quit© stable but it deteriorates easily in solution..
The
presence of air, traces of metals such as copper and iron,
and light, especially in th© presence of riboflavin,
affect it adversely (Hosenberg, '45).
Vitamin C is labile.
1?h© destruction of vitamin 0
by heating in a water solution ©an be measured and studied
quantitatively with reference to th© factors such as
length of exposure, degree of temperature, and hydrogen
ion concentration,
faking fee physical environment into
consideration, Delf states that th© antiscorbutic vitamin
tiftxen combined in the cells of th© cabbage leaf, ia more
resistant to heat destruction than when it is ia th© expressed juice.
Some understanding of the effect of heat
6
OR
the- antiscorbutic p-ropertioft of .teods ean fe© gain©i W
rmtmln^ aom^ ©f tho studies perfdmnftd cm eertala foo«S*
stuff9 (5d884 *20)»
Adooybltii aeia. is at or* atAble la ©u add tMm in a
nentraX mMtts® ma still idis .stable vftm th^ ft^dltin 4a
alkaline,.
By teata wife o»»g@ sad tmm Jtiice, Sol at and-
Frolleii dho«ed tfe© faet tMt tbd atntiaeorbutlc ^ittmia^.
vftim asaoeiatad witfe ®n aeid, is protoeted groa^Ljr twm th©
dettraettv^ effect of heat* fM@ wa^ omfinB^d bjr' tttamafoua
oba-©rtr#t0*
fomfeta^ eoKitainlns M#i. ageerb-ie ©eid, i# aa
e3?o.&ll@at wmmpl® of ®sir&©4 thjampstabili^r by its aold
reaotian*
It had been *$p0rt«& ©^ being atromgljr aati-
seorbtstie ©v®n aftos> it tern been fittfejeated to tb© oaiinliig
g3POo©sa..
HSS^SB.
and Zitvb hwro sbovsti that the antl*
seorbutio ,^©t©ncy of tomatoea &&$. deatro^d by dven dSl«^o
altell Ci®s0:* ■•*fiO)>
Sspyiaag oftwi r^jtolts to ooaaldovttble loaa of «»eorbi«
aeld*.
It h&0, b©©n #botm, boorave*, that fr©ab ailk d^ied
by fe*© jtast-Sotaakor prot©s,s fsstaifta an a^preciabX©
emoisiat of it« aattsoorbtttlo potency {S©s0#. *20}*
^alo^o^ ^f .^acoybi^ M|d
fb© faaetlon of a^eorbla aoid in ^# bo^y is as a
bydi?ogda tgaaaporte? la colltilsr respiration^, haviag
rororslbld OKidatiosa cuad redactioa e.ag>§t©ity.
Vitaadn 0 ia
gdUnfc fetsmtes is ■ tskongkt to jfottfttictt iss &mJtto^tiom with
ftaeorbltf aeid oxi^noft (oopper eeeabiaed tilth protoiu)*
Aa
Im^ortdAt part is placed by tfeo ©nzpa© tsfeiela -sozvdd as te©
aetiv* catalyst ia tto.© 0Ki<l^ti.<!>a'*3?©<la6tim s^ttim (Sfean©^
Ascorbic mM ae%iv©t©g a n^Eosbeip of mzym&j, ®mch ©©i
g&tfaejwl&jr ©jpgiaas©! papetia, ©gjyiaae^ catalas'©,. iatr«ft^
tppO'sinase^ aucleaiso, jpbotfjiiatas^i suecluic dtky^rogeaas®
and cytochrome Oiddaao.
Tiio fact that ascorbic acid
f«ftcti©a.s ©« a co^upa© o<r a pft?% of a cosisaym® fea^ btiin
obaervod*
fh# anoiaat of blood ©ttaras® waa fotmd to
fStafttit&fe* pvopdrtlmiftt^ly ^itii tb# «ttO<mt of ascorbic m:i&
^^misiaterad to a patiaiftt' s«iff03?ijag .frosa vitamin 0
60fl6l«n«y {Boasaberg, *4S)#
A$co>rbio acid also ro^lates th^ collolial ooaditloja
of iatarcellulaF tiasm© as wall as Itoactioaing in ifoe fox^a^
tlon of colloiiaal iatarcallmlar arabataacaa, ^aicb lacl'ada
feiosa of sloa3Ptilagai, desitia®, and tb© matrical of feona^8
md fur^tevBOr^^ tfe© collagen of all fibrous tlsstioa and
aoK^a-pitMlial eenmait atabstamcea*
During vltaaia 0
d@fiei@ncj fibrila or ooliagea sr© aot fo^aad noswally
(Soa©iib@r@f *4&)*
5ho fact that ^Itamija 0 is related to
calcium metabolism is siioim in tlio above pheBomsna v/rdcb
ar@ aiiailar to ttios® obaerred in. vitamin B defici^icy
(Soaenb^rg, »48).
dreetKte&4 Bn& S&rcl^ and Stag a&3 l©ate®» 1908^ foimi
tiliat an toerta^e Sn Intiak© ©f vitamin. 0 in&reaaed ^.#
s»©siatgi*ie0 of guinea |>iga t@ fe© iaftefclea ef a staadar^*'
Isdd Oijbtherla toxSn*
If viteMdto. © was lacking^ t&e
survival time of Hha gaiaea i^igs wat s&oiPfco&M 00 p&r cent.
®i$i»© wai « Qt»aatit&titr© r^atsKmeihip between vitfflK&n
Q intake' feed ^aaplmsnt tit^r In hsasen piaoma; -^i iafrro***
iia tii© irltiaaiB liat^^ aaerea^d ^a# ©iO!8spi©ss©sa,t la t&©
hlm& (<%m and eiiow* *$8)*
gbe eon^iioaifint itf t tS3wmd*
©omplomejat in tlb0 bledd s@3mm is to d9«l8Pogr bwothrift. an<3,
othef e@ii9-» A function ©f th# as^^rblo a6&6 eont^iat of
&a biooiS ]9ftrutt Is the iMiiittiMEMui«« of $ ;r*v9y#£$3U> wtida*
tioga^eduott^a fot^ntial ^sbibitad 1)^ th©" <J:«asiipi0iE©Kit!
(ioS'mib03pg.j *40)»
VitMslo C- it rolafe^d %o dejPbeaxjrAfeit? m©ta.boii©a,
Bspd^isMtaitMt b«ve jshdm ttet©t In presco^titio aad secNPtftttle
eonditloftS" ^aiaea ptg$ ^.avo «, iowor eapaeity
^OSP
m^talsoii-
jtiag etxgar ad d9t«-mliiad isqr m$&p- Wl&rm®® tmts (Sigal
Qtt4i 'King* *86).
Vitaaia 0 al.$o 9wm» %(* bo ooae^mei ia tfo© m©laboii$8i
of aaiao adide^ **-g** tgrspoftina aai |fc^a^lai9ain©# {Saaai
H^* X^vtoe efc al*#
f
30 im«t *4X)*
la vitro A^ariaants
bava iadtaatad i&mb aaiao acids ai?© ddbjrdrated by a^oorbia
0
Method a 0£ Aacorblc Acid A^ajg
fhls a|i9a7 Id teased on %h® amount of ascorMc ecl<3
mfiminitterod' and t^© oi©gi»©8 of p&eQVGr?
OP
©^ |>^v©ntioa
j^tJiOi of 'b.ioattay., ToatRg. v&mtX g®in®& p%$®* w^l'#iiiag
tvtttL $®0 to 960 gyaffiS' ara ssaimtatoe^ ©» a fear^al 4J©$ «aali>la
ia Adc^aAte la all. aufcriaata axoapt for vita»i«i. G*
c^e-
grouf of auiiffialS' uteifth ia f«4 tfe.# tea^al 4tot aloo'e*- aarvaa
as tb.a nagaMva o^fc^ol gromi)*
^tnhdr guiaaa pigs ar& fa4
diff©T®at amowtf of t&e ijatt^iaX to be toptog*
4t t)M»
atwl ©f tfea tosttng period ($ to 10 vrookf) tbo animals ar^
killed aaS antoiialai*
Bia dogroo of $rotootioK& agniftat
murvy iB<3ioat$a th® amount of asoorbio aoid in. the test
&*
f&o fturativ* matliO^ of bioaasa^ waa daaignad by
ia^ria amd feia aaaooiatas* At tbia toatbod yo^tilrot oon*
ai^arably t*»» tl«o aad ajatartal it is fj?e.f«©atly «8©<3.,
ML$n yotmg ^alnaa pig^ a^a feaft ooa a basal diet, devoid of
vltemia Gj, asm^ptositt of smrvj a^peaj? la ab©«t 10 to 14
days*
droufja of astimala ara gitren gra^ai ©mownta of teat
matariala and otbe? groups of ©fi.isiala are givaB. feamm
amoimfca of gwr© asoorbio &©!&♦ After 2% Says of auppl©*
meat feoding &%% mim&l8 «® atttopsi©*!.
ffe.© dmo^tnt of
asoorbio acid to the test material is ©Btimat®(3 bj oompar*
ing t^i© aeries of aaniisala which recelv©^ teoxm amouats. of
test aatoriala wi^i the animals Tshicfe received the know©
amountg of pure ascorhio acid {Be&sey, *38a)*
2. , Cb.emioal methods:
ffo® chemical methods ©re baaed upon th© fiact thsit th©
ascorbic acid reacts taith eertMn reegente giving epeeifie
roactions vshioh nsaj be meaeared by cblorimetry or titraticm.
fhe chemical method is mofe ra$id end acctarate than
bioasaay b«t probably no more specific
the general
raethoda are discussed, ae follows*
a*
Dye titration laetftiod for reduced ascorbic acid
(Panaer and Abt, '36) ♦
fhia nsethod was first introduced
by filluans and his associates and is based on the reduction of the dy©# g^S-diehloroghonolindojshenol* from a
blue color to a eelorlese eompound.." fh© follo^ng equa«>
tions show the reactions*
XI
M-
. 0=0-
I
10-C
II
ao-o
I
€=0
I
o
H-0—' + 01
I
0
HO -/\
,N
*V- v
+ 1-0—
I
01-
-,\ I.
cm
ph'inolipdiof^n©!
Tblu© in Qil£all#
s^a la aftld)
teaieato^
(colorless)
ebolofroaeetle a^ldUi have b©©a used t&r m© e^traotlea of
vitamin. 0 f^o© fooi 'Materials to ©fcaMXis©- th© vitjasin t&
SeXtition agdinat ais? oxiaatioji or
HOB©
of til© o^Mi^ing
siabstaaeos preaftat, aucb as «sll aaoiants of ©opp^r C'luaulfe
and Kiag, »®6}.
fh« roaetlon of vltamta C with ttb* d,^*® la
verj rapidj taking <»ily aboiat a sairmt© for titratieii
(Boaaey smd Sisag# <3S), oy 30 ssooaia for oolda?im0t|»y
(X^offlor an<a foating* H&),
fiae dye is a table in pov/der form b\2t slowly t^iangda if
in solutioa*
& small safiom&t of sbeaibato baffor* pi B.S^
is aided to the stoc^c aye solution f&loh should b© kept ia
the refrigerator in a dark bottle,
Fr©s& iye aeltatloas
should b© prepared weekly (Bessey eaad King, *35)*
12
b,
fedino titr&tlcoi method tor reiaeeia eaeorbic a.@id.
fh© Oofll i IMfe© soltaticm in 1.5 per o©at of po^^sslwm
iodide Is ttaed.
Starch Is ueod to Imdieat© th© ©nd point
of t&e titr^tion*
Saoh ail* of 0«.OX M iodlaei eolufclcn is
equivalent to 0*88 aig* of ascorble a eld (Bossoj md
. ©.
fh© g,4 ainitfophonjliiydrasin© method for total
aaoorbie ©eid,
fhla ast^iod was first ufied by Ho© ani
Kueth^r (1943) and thito modified, for th© aic^o ©na-l^i® bj
tmrj, tfOptoz aftd B®sa©y (1046 eM 194&}« • fM proeddcurd ©nd
r^aetloas ajr© diac\ias©d under th© aeetion on l^oeed^r^ g&nd
Ola^aalen of the Method of this paper*
®any etudiea ha^© beaa uaed to ©^al«at# ^ao ada^uaoy
of ©dsrfeain levela of aaeorbla a Old for good huaan nutrition*
It m®$ b© said that th© level a of aaaorble a aid say b© om*
aidered in three reapeete*
(1) in the eeadltim of body
aaturatien (Storvlak md E©wol£, *4B)# (d) at the modevate
level <aiDtith# ♦SS) and (3) at the minimum level ohieh la
luat aneugh to protest against aeturoy (Oothlln, '*54}*
generally» moat inveatigators uae one of the following
aietheda for avaltaating the atata of nvtvitien with re spaa t
to vitaaia
1.
GJT
©oneentration of aeoorbio aoid in the plaaiaa
13
■ %.<,
.$.
dally nri&^Tf exoretion of ft^tsorblo aeid
ufln&vy v®&pmii$® to a test dos© of ii$C03?bie
acid
4.
eoiafoinsfclcsi of the abotre.o
Keeootly the- relation, of e®otant# of aso0i?blc acid in w®&
6ell» m& plasssia (Ssrgeatg '47), a& wstX as tb.o aaeorble
ai§i<S eontent of white bl©o<S mlts &xsA platelets hair© also
been studied (Butler, Gushmsai and ffluttbctolmt■ *43* to^ry,
Bassej, Brook and Lopes, 'f46 and Sstsey et el,,. *47).
■ Abt, W&m®w m&i Bfjateta (1938) foirodl that blood .gdaan*
valuta leg© than 0.75 to 0,80 aig* per eent of, rediaeed
'^seorbic a old indicated a
subaottoal ^itaaia 0 int«ike»
treenberg, Ilnehart amd .Ituitek. (IdSd) coBiidered 0*7 to 0*9
mg# jper cent plaffiaa aseorbi® aeid as adequate but not o|)ti*
mal*
Soith <1€9$) #^g@estfd -tbat th.0 degree ©f aaturatiom
of aeoorbic sold in blood plasma aay be classified ae
followa?
foor conditicm
0,00 to 0*4 mg* per ceafe
'of ascorbie acid
Hoderat© ©oaditiOB
0*4 to 0*8 mg* per ceat
ascorbic acid
Tfery good eonditiesa
0,8 to 1*2 ®g* per cent
aueorbic acid
B^cellejat conditioa
l,i or above mg, per cent
©fcorbie acld*-
Salli aud her asaooiatea (1939) reported tliat a plasaia
eoneeBtratifia of ascorbic acid of 1*0 rag* per ceat could be
24
eottsidered mat ti© $^bj®tt Md m opt&swa Sally Intake of
Urinary e^e^fei^a sis© Ms ^©aa ©(oniiaer©!! as an to^l*
eatioa of me state of aseorbio a:©ia nttferitlOB-in the bofty*
If the body 10 saot in « tatwrat^a 0QB<3iti©m9 in© ©aot"aiti<m
of ©$©orbie sold in th*. urine is saalX (Boas «nd Berj-jawi^s
Hi,)*
XR atibjects ntios© tlssaw©a tsr^r©
EO%
£|6tut&t«d with
aseorbi© eteid before th® beglmilng of ©«|5®^iis^n%$l period^
a aaiif i&t«ac« of X00 mg« of ©aoorble aoiS pftr -dtey r©'ault-©4
In the wim^ «xerdtlon of no ia©r^ feau an Averftgo of
IS mg* of Q«6QPblo moid po* day (fiRlli^ ITit^maa a^ SMyry,
fe© rfilatioiiship of-thie wyiaary exerotlon of aaoorblo
«eid «aid the'«on«^itr«tton of as©o^b£o- soli in bl0©«3 pl^nM
to tiAawtd g&tnratio& ha© bewi r<&porto4 by.©th©2? wovkere
{8»%a*vf Bsuok «Bia Stopviok* W).
m 1940 Stonrl^k and.
Bauok m^iixg Adult sabjdota studied th© urlnd^y ©xeretloa
and plfi0iaa eonficnatoatioa of tsoorblo Aoid during poriods on
oontrollei tseorbie mM Intake*
fh© bod^r Ha«iuaa of t&o
Aahj'ootd mm praTieusXy satwrstod with Yitaala c ©ad t^©n
atftlntainad ©n © haaal dtot low la Titamin © a&d cuippia*
aentod with ^arloua latrals of ayataaetlc aaeorbi^ ^©ld.
binary imltaos w^ro detaswliiad dally «n g4?h0ti* 'ai$$iBi&B9.
A ateaaddrd tost dos© waa* tas^d at tbo ©nd of ®a©h period to
find "out tho stata of tlaatae reaerv*s«
Finally, tha
15
amount of ascorbic aeid which would maintain the tissues in
a fully saturated state was detemlned.
The results showed
that individuals varied in their response to various levels
of ageorble acid intake,
fhis individual variation in
reaponae was eonfimed by I*ewis* storvlck and Mmek (1043)
and by Klliie and Bheart (1044) ►
16
im mMPom of
TBIB
i&vBaiism®zm
Humerous studies to dtteymine'the requirement of
aseorfele acid in adalta sad in children hare been ma^e bn%
studies on the requirement of cscorblc acid in adolescent
children have been made only recently, (stoxviotc et al.,
H7)t i.e*, in the last three yeara.
Sa most ease** isfeere
blood studies liave been made, plasma is the fraction of the
blood which has been analysed most coimonly for ascorbic
acid.
Both dehydroascorblc acid and ascorbic acid in reduced
form are biologically active (litchell, '46).
Experiments
have shown that dehydroascorblc acid is changed to the reduced form In the animal body (Johnson and Zilva, '34).
Four adolescent boys served as subjects during a 30day experiment divided Into three periods of 10 days each.
During the first period they received 200 mg. crystalline
ascorbic acid1 per day in addition to the ascorbic acid in
food| the second period they received the Recommended
Allowance of the National Research Council, or a daily
supplement of 80 mg. crystalline ascorbic acid plus the
20 mg* of ascorbic acid In the controlled diet, and during
1 Crystalline ascorbic acid was obtained from Merck and
Company, Hahway, lew Jersey.
17
the third p&rio& they reftdlvoti a $®t%j supplement ©f 10 mg*
less than tno noaon^^i'led iaiocance or tlio llaticnal
i£@'8ea?<& Cotmell,. #3? a dail^r sufplemisnt of fO ag# ©f
etfjwtaXlin^ aso«rbl« aeld plus 2t »g» of .^dicorbio 'a«l4 in
food,
• $h0 purpose of tht9 stuady wsiS'i
1*
to &m®lQp && t@<$mim® tor th© d^tewaiuation
of aennua aaodrbi©' aeld vising the alerd*ia@thd&
of Lowj»y8 XiOj>©'Z 4U»S-Be08«y (1945) I
2*
to aak© daily 49,&eroiJtofttl«nd of %&ft total «.s.eos?!ble
aoid oontent la the a arum of adolosoeat ho^ra
smlatateoi ©n diets •shieh w©^© gnalysdd for
3«
to emp®F9 th© (feily d©t<&»liastioas of total
ftsoorbie ft»td is th@ mtm. vdth t&€» valnea obt@i8:#d fox4 pS)d«©«S avoovblft &*ia ift tho plamiei
4#
to oompaf® th© relati^^ @ff®et:if©n#s# of th<l
thif©© levels of a#e©i%i§ aotd tetuMo ia malm*
ta&aing th© oone^atration of afoorbio aotd iri th©
IS
eam-gt n.x
EXPEHIEENTAL
four Golleg© f^os&aaa hofti, 18 y^^ra old* flowed ««
•Stt1fe>|<ects la tola'Investigation*
fh®f wev® &pp&?mt%i$
nomaX and la' good -pbysieel ^mdltlon' a^di odrfl^d on tMij?
UBtttl se^ooi tiOFle ^urlag th© time of tM» stti%«.
Age,
hoigfet, weight, weight r&n@© aad- wfrlgbt varifitloft for ©ash
•ex$&@j?im@*it;ai' aubl^et a-r© tixem as if allows.t
Subl^ot
Ag? . felgiife
•to*
-fllfeatt weight
"' ■j,ib;u • fc.i#
a@
73.5
xad
. is
73*8
w.r*
2,®
B.E*
3,$
$♦0*
D*^'. 0
:
variation
te
iis:"""
18g
m 1$S *1@0
m 178 -18S
7
m.®
18©
59
1261-130
3^4
70*S '
161
69
l46|-lSSi
7
@
All aab^ootd were well during &© on tiro stwSy oxeopt
p.t*^. w^o v-repoaftod t&at. h© vat not fooling woll m tsho
sceond day of the saturation period andt stated that ho had
dlarx-hca and cold ayaptoms.
Since he did not Oat all of
fcho foods aorvoa at a upper time, he craa gi^on 30 atg* of
er^aitallXno saoorMe aoifi to bring his total aseortoic in*
take for th© day up to th© amount r©ooiv©4 by the other
10
sulbjeets* Be had mQ&v&rGd fTom Ms aymptem te^ ^© next
<Say,
Fro® tii© beglimtog to th© end of tl3Ba 50*<!&3r expoFl*
meatal. atuiSy, f »G«» ©♦£■*, W*r. and D.g. gaiia;$a ^;>. 4.5^ 8*$
aad &.$ poufids, rdapotstl^elf *
Plan of Expoite-AQnt
TkQ 30-day ©xporliBent iai5t©4 trm Jaattajpy $1 to Isrefct
X# t94B aiaa «naa divided iato tfer©6 j>®pioda of tea days
eaeh*
ffeo firfit period was tli<s» saturation period* the
aeccnd period was designed to test the National Eeaaarch
Council recoiesionded allovjanco of 100 rag. of ascorbic acid
per day for |8«y©ar old tjoys, mad t&e third period sras to
test the adequacy of 10 mg« lose than National Research
Coiaaeil i?eeo®iffieadatl(m*
For the fir at 10«*day period a
finapplemeat of £00 mg* of erystallla© ascorbic moid mn
given to each subject in addition to what he received in hie
food.
During the seocmd 10-day period* the ascortoic acid
from food was restrleted to SO mg» per' day and asa 80 «a§»
aupplement of crystalline ascorbic acid was givess dally♦
In the third period the diet captained the same amount of
ascorbic add as in the second per-lod, and a aupplemGnt of
70 ag, crystalHiie ascorhlc acid was ^Lvea daily*
the
blood v/aa taken every day before breakfast and the seruia
wad analysed for total ascorbic acid according to the
20
Sbs aiiljj@©Ua vei$i66 themselvoa ^ei*y ^ay befera
Goatrdl of Ascorfei©
A^I^
All tood oae vsl^iod aad fe© ase©j?bi<i iacid ©©atsat
was woigji^i aad ijasedtiafcaXy pwt la tho '1 per e@a% »©%©*»
^bdspborlo ©ei<3 soiafcloji ($0 $3i?asia9 of food ismpl* to 100
ml, 1 pfcs? cent Bi©t6plios|iiii0f,io aoid) In -a. 3ia««i& ja^*
fho
ooat^iita of tM© |ar ^w>r« ml*o4 utifog a Warlag 81<Mitler anl
w«»i>© flltarod*
®&0- asoopfetO' e«ii4 w&* do.t^painod tsaliig
aodlum 2,6 dichlorobenaenonoindopftienol (dye).
Aliquots
war© road ©a an l^alya Ridteeieotvla OoX^rlaoter with
1 Fooda wayo analyzed for asoorble a old by Bath ^off&y
aooording to thomathod of LoettXev aad Pomtlmg {Wifelf*
21
S®fc©minatS.on of ^seorble Aold in BXood Sanaa
Equipmentt
1*
Constriction nieropipettes Cbsnd type), 3.0,
&Q9 40, anci 50 ©..Jstru V<BT® aad® by using glass
tubing, standard wall
n
pjrexm glass of 4 HESW
outaid© dioodter and calibrated % filling to
tli© eonstrietioa froa. a 0.2 wl* graduated
pipette*
fb© pipettes wore eleenedl %?ith dia-
tilled wat©!9, 95 p®? etnt aleohol and ether
after aacfe sampling using a sueti«Hi pump,
&am©times a .Haemo-Sol solution was used for
©leaning pipettes if they confeained serua
preelpitatee*
2*
!&© 4-.ineh pieces of glass tubings stendiard
wall
n
pgTexm glass of 3 sm* outside diameter
were cleaned by boiling trfLth Ifl IflOg and
rinsed with water and distilled viater and
dried for eollection of blood samples.
3.
Pyseal for sealing the blood sample tubes.
4.
A Beekman fipeetrofhotemeter fitted with apeeial
diapferagra and equipped ^ith quarts miero
©uvettes, 2..5 aaa* x 35 SM. t© peimit the us© ©f
saall ^liquid volumes.
Diapbra^a and ewrettes
are obta,in©ble from tb.© Pyroeell Manufacturing
Company, 207 last 84th. Street, lew fork City.
132
The ©wottea wer$ always kept ia a defiait®
order and positiorij, and ware cl©an©c3 by rinding wit3a. <aistlll©<S wafc©r, 95 p©r e©nt aleohoi and ethor after ©aeh ssries of samples
had been road.
5.
S©rologi©al tubes, 6 x 50 sam, ®.g*# Kirabl©,
lo. 4S060.
6.
Alimin«m racks for .small tubes, 6 in* x 6 in. x
1 in. eontaining 100 holes of 7 mm* diam©t©r
(Sorthsm Tool and Inatruaani Co., 164-21
B©rtli#3?n Blvd., Plusiilng*. lew Tork).
7»
So. l-A Vial rubber stoppers w©ro used for
presenting evaporatloa of the -small samples
(West and Company, Ifeoenixville,. J'enn,).
8*
Oentrifug©..
<£hM Xatexnatlo&al Cllnieal Centrl*
fug© with regulep- aiero heads. Ho, 11758.
9.
An incubator vfoieh ©ould b© set at 38° 0«
10*
Foreelain pipette holder*
11.
Blade (Bard Parker)*
IS*
Eand@@*to©l {bus&er) (Ohieago Hieel and Manufaeturing Co-*)*
Eeagents j31.
5 p@r eent thiourea in redistilled water
1 All reagents wiere kept in the refrigerator when not in
use.
S3
2*
0.6 par cent CuSO^SJIgO in yadtstillaa water*
5.
0*8 per eent <aiatti»oi&©nyl3b,ydraa3jQ© In 10 H
HgSO^*
4*
Centrifuge before uslag*
Thiourea copper auifate-ainitrophenylhydrazlne
reagent*
Combine 1 rolum© reagent 1, 1 volume
reagent 2 and 20 voluraea reagent 3 to prepare
reagent 4.
Reagent 4 is stable for at least
one week if kept at 4° C. and should b©
oentrifwged before ue© wnlegs it ia crystalclear.
S*
© per cent trlehloroaeetie acid,
this was made
dally using redistilled water*
6.
08 per cent %$04 (70 ml. of eancentrated %S04
plus 30 ml. of redistilled water)*
7.
standard ascorbic acid solutions:
100 mg* of crystalline ascorbic acid were
dissolved and diluted to 100 ml* with 5 per
cent trichloroacetic add and 10 ml* of
this solution were diluted to 100 ml. with
red!stilled water and then further diluted
as follow©j
a.
4 ml* to 100 ml. with redistilled
HgO
^ 0*4 rag. %
24
b.
10 sal, to 100 ml. with redistilled
SgO ^^ 1.0 ag* %
v..
16 ml. to 100 ml. with redistilled
EgO =a= 1.6 mg» %
d.
20 ml. to 100 ill* with redistilled
Hg0 === 2*0 tag* ^
10
6«MU
-siimpl©:Q of daoh of the abotf® w©r©
aoasmred into 6 ^ ©0 »# tiibea with a .ton*
at2*iOtiari pipett© ©Bd 40 e#wu S par o©nt
triohloroa^^'tie ■aoid war© addad with a eoaatriotisn pi|i0tto.
©ad^ro.)
{sm Metloid on pro*--
Ba.t©na.ia&ti«Kis of ascorbic, aaid
for oaiph dilution mvo mada in triplicate.
8,
Blnnkoj
10 <j.aiia. radiatllled water war® measured
into 6 x SO mm» tubes and 40 cmm. 5 per
cent trichloroaceti© a©id war© added.
section on procedure*)
(See
fhis ?;aa also done
in •triplicate.
Procedure.
The blood was taken in the early saomlng before breakfaat by finger puncture. Two 4*'inoh lengths of chemically
clean glass tubing t?ere filled two^thirda full with blood
from each subject,
the blood was allowed to coagulate by
resting the tubes on the porcelain rack for about five
minut^is.
One ©ad of eao& ttibe was the© aealed witb. pjs^al
aact the otoer endi was capped with a small (Be* 1*A) rubber
stopper,
fb© tubes were e®ntrifug®d witto. tlie pyseal ends
do-em for 10 minutea at full spaed,
ffee tube was out witai
a amall file Ju$t a little bit abov© tbe layer of the eella
In order to separate blood eeilg ©sad serum.
Some © & 50 «t. tubes were placed in an alualnuia raok
and 40 e«aja. $ per oent triohloroaoetio acid Kstre traa.«* .
ferred to eaob. tub©! 3.® 6>»m» of s^rum were tftsa added and
tb® eonteata jal&ed hf tapping with a buisaer.
^o tubes
were eapped wltb so*- 1*A rubber vial stoppers and 'cen-tri*
fU'ged for &0 minutes at full *$$*&»
& $0 e*mm. aliquot of
tb© aupernatant was transferred to ©n@tSaer 6 at SO mm*, tube,
and 10 cspft. of the thlourea copper fiulfate-dinitropbenylhydrafclae r^ageat (reagent 4) tsw* added,
teagent 4 vaa
also added to tubes containing 30 c.wm* aliquots of standard and blank solutions*
5be mmm samples^ atandards mid
blanks were don© in triplicate*
All tubes were eapped as
before and tapped with tb.e twusfter*
•Jbey were then pat in
the incubator for 4 hours at 38^0* At the end of tb©
Incubation period they wr© tafeen out snd tesediately
chlll©d in ie© mter and 50 ccaa. of ic© cold ©5 p&r cent
IgSO^ were added*
Becaue© of tho viscosity of the acid,
^© pipette tsas emptied slowly.
ala®d with, the ua© of the busser.
Th® contents were again
After standing for' frora
20
30 minutes to 3 hours at room tomSKraturft, &© tub©® ware
again mixed by tapping vtLth tho flng©r# oad th© eoateata
transferred to cuvettes using e ecustrietloci pipette*
tEbe
light absorptloa ©©a ^©surod at 820 aps, tjltto a slit width
0*1 sm* In the Beetemaa speetrophotometer*
flae water blanks \?©r© read before mj of tto© saiaples
were read In order to detect nftiether or not the euvettee
were ©le©n*
Ihen the ressgent blanfe$, standards and serum
samples were read.
The serum ascorbic sold determinations
were made dally as mom as the blood aaapXee had. been taken*
Ike reeding of optleal density mlnua the blank reading
gave i&e oorrected optloal denalty reading* fhe aveTage
of thre© readings was used to read the tag* per eent gacorble
acid from the averag# etandard ouripe.
5he average values
for the standard curves1 are tafoom in Tables XA* IB, «ad
XC«
®i# currea- with saean devletiene are ehoun In Charts
Xkt IB, and jc*
The following Is a a«mple record of one
day*s anttlyseei
1 Curves were calculated for each pipette*
27
Headings for standard solutions?
f
width
ma.
Sample
Water
Blank
1
0.1
dpii'c al Pen a Ity aaadlnga
Sample
Sample -Island
Average
1
2
2
3
2
1
12
2
10
3
11
-
0.4 mg. % 1
83
12
41
2
56
10
46
3
52
11
41
1.0 mg. % 1
116
12
104
2
114
10
104
3
111
11
100
1.6 mg. $ 1
175
12
163
2
176
10
166
3
178
11
167
2.0 mg. $ 1
214
12
202
2
212
10
202
3
219
11
208
43
103
165
204
28
Headings for serum ascorMe acidj
Ascorbic
aeid in
Optloal Density Headings
Subjects
Slit
Width
Sample Blank
Sample-Blank Average
' ■'"Wo""'
sag. %
138
12
126
2 •
130
10
120
3
138
11
127
p.B* 1
131
12
119
2
134
10
124
3
130
11
119
W.P, 1
13S
12
123
2
135
10
125
3
131
11
120
p.H. 1
123
12
111
2
122
10
112
3
124
11
113
T.C. 1
O.l
124
1.21
121
1.18
123
1.20
112
1.09
1 Using th© average optical density (for reading - blank,
YJhich isi Do) on© obtains th© valtaes for rag. $ ascorbic
. acid in 3®Tvm hj reading th© valwss off standard ctar?e
... on Chart 10.
29
2fi.BLE Ik
The optieal d@»8iti0s^ of standard aseorBic a@i'd
selutlcme and their moaa deTi&tiaad
(lO G+WB*- plpett© Wok, 1)
joao'entriation* e'sprWaed in mg* fe of*
acid in solutioae
1
2
5
i*
5
6
7
8
9
10
9
11
13
11
8
,9
13
13
21
22
21
2®
21
go
23
22
n
39
ItB
it3
47
Uh
h5
3©
39
JL
fotal
170
21
21*21= 0
22*21= 1
21*21= 0
20*21=*!
21*21= 0
20»21=*1
13*11= 2 23*21= 2
13*11= 2 22*21= 1
■u
Si
o
0
100
94
96
ill
106
111
100
561
106
1016
102
*■#■
11
9*11=*2
11-lfe: 0
13*11= 2
11-11= 0
8-li=»3
93
99
1*0
38
12'
484^3= 5
£34*5=0
47*43=4
44*43=1
43-43=0
38*43^5
39*43 =4-^
52*43=9
4o*43=*3
38*43=*5
42*43=*!
48*43= 5
206
211
206
196
204
201
'189
1614.
168
161
II4S
155
147
155
147
174
I65
160
93*102=*9
99*102=*3
1OO*102=*2
94*102=*8
96*102=#6
111*102=9
106*102=4
111*102= 9
100*102=**2
106*102= 4
168
2072
159
164-159= 5
168*159= 9
161*159= 2
148*159=- 11
155*159=- •4
1^7*159=^ is
155*159^ ^ 4
147*159=* 42
174-159= 15
165*159= 6
160*159= 1
160*159= 1
168*159= 9
*y=»5*6Q
l|=*6*38
229
217
201
201
214
'8661
205
206*205=
211*205=
206*205=
196*205=*
204*205=*
201*205=*
189*205=*l6
186*205=*19
229*205= as*
217*205=12
201*205=>* 4
^>1*205=* 4
214*205= 9
1 Gorr©et©d hf sub'teaotlng tho reading for the blarak from th©
reading for the samplss
31
TABLE IB
i
Th© optical densities of standard ssoorbie aoid
aolutioos and their msaa deviations
(10 ©«Bim» pip©tt© So.# 2)
Sample
1
g
5
h
5
6
1
Totel
Mean
D&tri&tioa
from th©
mean
Meaa
dsviation
from th«
mean
Conoeatration, expressed in jag* %$ of
aooorblo aoid in solutioaas
1.0
1.6
Q.k
2*0
ho
k6
107
102
106
163
165
164
192
209
200
I4S
101
160
201
37
M
95
16©
198
102
160
206
163
203
1135
162
12*09
201
1*1
U3
^
105
718
103
UWt8= »1
1^0*1*2= «2
146-1*2= 4
kB*h£= 6
37-42= *5
ia*i42=*i
43*42= 1
107*103= h
102*103= *i
106*103= 3
101*103= »2
95-103 =*8
102*.103 = *1
105-103= 2
296
20
y= +2.86
21
■y=
'
±3.0
163*162= 1
165-162= 3
16^162= 2
160-162 = *2
166*162 = *8
160*162= *2
163*162 = 1
T^=
41.86
192*201= *9
209*201= 8
200-201= -1
198*201= *3
201*201= 0
206*201= 5
203*201= 2
tr-
±4*0
,
1 Corrected by aubtractisg the readiag for tha blaak from the reading
for the sasple*
38
i
the optical densities of standard asoorbi© aold
solutions asd their mean dsviatioas
(10 e«,m« pip©tt® Io# 3)
1
jSampl®
Gon<3©ntratlon9 estprsseod la s!g» ^ of
asoorbie aoid in solutlojas
1*0
.
1*6
.... G*k
2*0
Bat®
1 ' 2/21
2 ' 2/22
3
2/25
h
2/3k
5
6
7
8
' 2/25
3/28
2/29
3/1
MOQJS
Deviation
from th®
mean
pleaa
doxriatiott
rrom the
meas
42
h3
k3
46.
.14
163
163
I65
169
165
159
42
44
104
101
103
106
101
100
103
103
164
207
202
204
210
203
201
209
208
42.75
43
102*62
103
164*12
164
205.50
206
la
42*43= i
43*43= o
43*43= o
46-43= 3
4l-43=*»2
41*43= *2
42*43=-l
44-43= i
1to=±l«5
165
104*103= 1
101-103= *2
103-103= o
106*403= 3
101-103 = «2
100*103=»3
103-103= o
105-103 = 0
163*164- »1
165-164= 1
169-164= 5
165*1614- 1
159-164= *5
165-164= 1
164-164= 0
207-206= 1
202-206= 44
204-206 =«2
210*206= 4
203-206=-3
201-206=*5
209*206= 3
208-206= 2
11
., ,
1-=
±U4
f = ±U9
£=±5
163*164= *1
1 Correet^d by subtracting th© reading for the blatafe from the reading
for th© samplo*
I77^
......r-.r
TT-
t
!
'
■
MC: :i...ti.
iTAi^DARlp'-tfeE FQR 3cbBBl!c]AJClb;:j
ICW ART
::.|:
S5
ffhe Discussion of the Metaaod
ThQ method of I»owry, I.opez and Besaey ia more satiafactory for studies on nutritional status than the earlier
met&tods alnee only a very small quantity of blood easily
obtainable by finger puncture* is needed*
For example^ the
method of Butler, Cuahaan and Maekaehian (1943) requires at
least 0*1 ml* of serum eompsred with 0.01 ml. needed for
the method of fcowry, topea and Begsey (1945).
Wx&n the 2,4 dlnitrophenylhydrasine derivative of de*
hydroaacorbic acid is added to 65 per cent sulfuri© acid a
reddish colored product is formed which is absorbed at 490
to S3® ap according to the study of the absorption curves
with ascorbic acid solutions of three different concentre*
tions (Chart 11}*
the jproportloaality of the color oh*
tained in this reaction is in agreement with Beer's law in
the ranges used (Hoe and Kuether, »43)*
Ihen methylene blue or dichloro^henolindophenol (dl*
chlorobenzenoneindophenol) are used in the determination of
ascorbl© acid, only the reduced fom is ffieasured*
However,
in the methods using 2,4 dinitroj&enylhydraalne, all of the
aaeorbic acid is determined in the fom of dehydroaaeorbie
acid since the ascorbic acid ttiieh is present in the re*
duced form is oxidiaed to fee dehydro fom before the
analysis is complete.
It has been found that dehydro-
ascorbic aeid (or a derivative) ©hlch reacts with dinitro*
36
pfoenyl&ycira&iia© is remarkably stable la serum after th©
ad<aitic«i of triehloroacefcio aeid, ttxether or not the
solution la separates fro® the protein precipitate (Iiowry^
Lopez and Bessey, *45)»
thlourea is uae<i to produce a mildly reducing medium
v&lgh prevents interference due to oxidanta.
Oolor is
produced by adding 6& per cent aulfuric acid and it is
quite stable*
fhere is no change upon standing for 40
minutes (Roe and Ituether, '43),
m
CH)«r»Tf It ht^i
l::rr
L xbao
!
■
I.-..:
ASCORBIC AdiD* AB^OHfTJOlil JCURYESJ
r • i:
:
■•':
•:!■
_illLl
'•
l ..:...
S
£rB\:
I :•:!.:
h2& 4&%i-STAWDARO ^Ot,W-TI««
jllioo:.
!
*:
■
xoa J
pL^oo.
iiEhffilH
la-aiflfci«Hi:iM^iMain::trfl:M
CmPTBR IV
38
RESULTS AND DISGUSSIOH
The dally aerum total ascorbic acid values of four
adolescent boys during the whole experiment period are
shovm in Table 111 and Chart III.
These subjects showed
daily fluctuations in concentration of ascorbic acid in
the serum at all levels of Intakes.
This was also obser-
ved by storvick and Hauck (1942) in their studies on the
concentration of ascorbic acid of the plasma in adult subjects.
The responses to different levels of ascorbic acid
intake showed variation among individuals*
For example,
the mean values for serum total ascorbic acid in the saturation period of the subjects T.C., JD.B., W.P., and O.K.
were 1.29, 1.27, 1.56 and 1.49 mg. per cent respectively.
During the period isshen the total dally intake of ascorbic
acid was 100 mg., the serum total ascorbic acid values were
1.23, 1.10, 1.20 and 1.08 mg. per cent and when the subjects
received 90 ng. of ascorbic acid per day, the serum ascorbic
acid values were 1.25, 1.19, 1.16 and 1.22 rag. per cent
respectively.
A change in the ascorbic acid intake did not immediately affect the serum ascorbic acid value.
A few days were
required by the subjects to adjust to a new level.
Storvick and Hauck (1942), whose subjects received large
amounts of ascorbic acid during the pro-experimental period
3©
of intake, found that most of their caaea needed 2 or 3
days for the adjustment to a new level.
In this study, in
Taihich the subject® were not saturated with ascorbic acid
during a pre-experlsjental periods, £&® results of the first
five days were not included in the final statistical analy*
sis.
According to Holmes9 Cullen and Halson (1941) and
Storvick and Sauck (1942) the mean value of a number of
plasma ascorbic acid determinations 1© teore indieativ© of
an individual's state of nutrition with respect to ascorbic
acid than is a single determination,
HhB means of the in-
take of ascorbic aci$ and the means of serum total ascorbic
acid values for the three experimental periods are ahom in
Table It,
fhe mean serum ascorbic acid values for all four
subjects for the last 5 days during the saturation period
was 1,40 mg, per cent and ranged from 1,27 to 1.56 mg, per
cent,
This agrees with Faulkner and Taylor (1938) and
Salli et al, (1939) who reported that the renal threshold
for ascorbic acid was from 1.3 to 1.4 mg* per 100 cc.
Dur-
ing the period when the subjects received the National Research Councilta Recommended Allowance of ascorbic acid,
i.e., 100 mg. daily, the mean serum ascorbic acid value was
1.15 mg. per cent and ranged from 1.08 to 1.23 mg. per
cent.
1/hen the subjects received 10 mg. less than the He-
commended Allowance of the National Research Council# or
90 mg. daily, the mean serum ascorbic acid content was 1.20
mg. per cent and ranged from l»l6 to 1*2$ mg. per cent.
The differences between the mean aerum ascorbic acid
values for the saturation period and the period rahen the
subjects received the Recoamended Allowance of the Mational
Research Council were statistically significant.
However,
there was not a statistically significant difference between the mean serum ascorbic acid values of the period
when the subjects received the Reeosmended Allowance of th©
Mational Research council and the period when they received
10 rag. less than the Reconsaended Allowance of the National
Research Council.
Therefore, one may coaclud© that for the
subjects in this study, a daily supplement of 90 mg. of
ascorbic acid is as satisfactory as the 100 mg. supplement.
Th© standard deviations of the means for seruai ascorbic
were not significantly greater during the higher intake
periods (Table II).
This was also observed by Storvick and
Hauck (19^2) in studies on plasma.
In general, fluctuations in senna ascorbic acid values
were similar to and in the same direction as those observed
in the ascorbic acid of the plasma;■ however, that was not
always the case as ean be seen in Chart IV A, B, C, D and &
One of the reasons why the ascorbic acid values for the
serua are higher than those for the plasm is because of
the difference which is Inherent in the method, i.e., total
ascorbic acid was determined in the serum and only the
41
reduced aseorbic acid is determined in the plasma,
Whether
there is a signifioant difforenc© in til© ascorbie acid content of aerisa and plasma would have to be d©t<sraiin©d by
applying the sam© method to both blood fractions*
Th@ mean differene© between s©rum total ascorbic acid
and plasma reduced ascorbic acid content was 0,41 ag. per
e®nt»
From tia© data obtained on© could predict statisti-
cally that tfa© difforenc© between th© serwai total ascorbic
acid content and thfc plasma reduced ascorbic acid content
would Tary from 0,37 to 0,44 ag. per cent in 95 per cent of
the cases (statistical analysis in fable V).
As tested by
analysis of variance these differences were independent of
level of intake of ascorbic acid or individual variation
among subjects.
TkRIM II
fh© msaa aseorbio sold ©©stoat of tfc© seruoi ©ad the ©easa iatafcs of aseorMo
acid for fouy adol©s©©at boys on thre® i©ir©ls of ©soorbio aoid iatake
?©rlod J
Satttratioa period
Sublet
:©aa A.A-*
Intake
r
"Period' II
M0 reoojamoaded aileraRa©^
li'gfeeot
la
*M©a» A»AV
"^eaa A»A«
^Qtioi'ltt "
10 wg-, less than
aa A-.A*
Intia&e
Intak©
Valea
m* %
T.O*
278:1 fi5
1*89
±0.17
1.55
1004 0J4,
(99^101)
:O.K.
g70ig9
(2^3-320)
1*27
■±0*15
1*246
100i044
(99«rl01)
1*2
looio*!*
1.7
?*?-.
0.H.
1:56
±0#10
:
lJi9
+0.10
■
1*7®
{99*ibi)
U55
l.k
(99*101).
1*23
±0*07
90 i 0^6
(87*92)
3...10
90 ±0,6
(87«92)
1*1
±0-*©6
1*19
40.09
l«rS0
±0.15
90 i 0*6
(87-92)
1*5
1-.16
40,15
1*08
9© ±0.6
(87-9S)
1.3
-l-o*io
1*85
±0^04
use
4o,0g
1 S'bandas'd doiriatioBS desigmted-by ± sigs» •
2 feag© of values espressed by numbars ia pa5r©n#!iss©s»
so
M
m
TO
r-l
a
•tl ©
o
©
»
o ■»
1
fa* Pi
«•
O
a
^ ea
fr4
•a
p o
til ©
^
to
at
CO
©3
M
I3H
^,
»
ss.
*
^3.
49
#•^1 ('"Q p^
#■■♦:•
«
#
f*« l*» iH »< M IH
UMOcO ONQN O CQ-^JtS^t^
• ■■»■» • •«
co J^-^J £*- O pr» t- irs-4 ©v 00
«
e
«
p4 f~t f!4 iH
*
•» *
4
•
*
»
«
»-J fj
-^ ^O ©N BC? Q OO ri. CF. O r^
e • \»
'» ■ *
a
f• *
r4
*
♦
#
»
H P* »~i
•
•
*
•
fH iH »-«
•
^>^g>eN© eavo osgj mvo
»• »
• • *
©ONr-F-tot~-p-©e-r~
p*4
o
•
•
•
*
0
»
o
r4 e=4 «~4 *>>^ »«<
o
*
*
*
0
e
-it W. *-) tH H o ^4 cy <w H
••
♦
♦«
w\
OD-d-ONirt fTvO ©CDJACO
1
8$0£g$£$£«r
p4 »-* «H »«4
f^i f^i fH p^
GO opa> IAHLONN^
• • • • •
l*H '#-4 ft t4 f*t r<i «*■» rt »*5 e^ fH
«AW>3US&J
a
•
•
•
>
■■•
•"#
*
«
f
«
e*^ tf*^
*
ir*4
«
aSJt mtovpcsctf cy MNCVJ f-»
* * • 4 * 1 > • • »
^ O^G? ^.^ ON ON (3\ (JN
,* •
'» » *
PH
^vo ^-aftA^QD GNe-~Na
t^^tw in3
© **» tAe^e-* cvs f-vQ ©NSQ ON
mtcsRSK> as«vi
J1AN0
» t
9 ©«>a>NQ trv
.* ' # *. » * • * « • • *
|H !*•• ©
,
~ ^Nl■ t?H
•
fS^^0
>
• * * t-i «H
»>4
«
.« * •
* • • • •
H^ **"♦ f^ l-^ f*^
•
•
«
»
#
«
e •
*
«
-O^'©
«
«
PfN_!j KN-4 tf\@ © fTNW CV» JA
»-i cvt tA*^ trvvo s^ 00 ON ©
or or of
r* jrt jrt jH jr« ^4 ,?"* J-« ^* ^1
s
Hi
M
f^s
CO r**
«s
*
♦.
'♦
ON vO
C^^^eU CVi CM CJ C\i €\> WN
^4 ir*4 #*4 ^4 (p«4 i^J tf^ ^4 f*^ r^i
i*4
*&$!?$£$$»£
Tt^sM^R^ifia
*»
O r-J r-4 O N>»^ *«« r^ O
S^ to mso tf\e*-co co b-
vO O r^ C—*«» e~-vO ft 4AO
CV!\0 p-CQ IAGO r* f~fC\e-
• •»♦♦••♦••
r< rt rt t**
!»« »-* rt fH
i<-} ►-» ■ 0 C—^ E~- SIN pr\
8lSM^PlS8fi
* ■*
M^^^^M
f^W^^ KN-d-KSPfVvS ITv
t*-1~~ f-» t- e*- c* ^ft^8^
• ••*•*«
« *
6R.
s&
a«i
es.
r^
en
._„
—
u%
1 UJ
Pi
<
u
04
o
w
*J:
<
..—
z
a
u
<
u
o
u
O
ii
1
■3.
::::
< '.I'.
< tu ::::*
> ' '.*'
III
-1
2
<l
21 2!
LU
o
■i>. u
_J H
■
! I.
::::
< LL ::::
a
<
N
w
- ——
—
-~'
a
to
s
V
jt
/
•
■ jj
xr*'
/
s
■/■■:
/
I
\
^
s
J
/
/
N
S
\
A
/
•'
"> ->.
• y
-«s
::..,
/
v.:'
1
1
1
1
1
J
>
V
■*^:.
::• <
•r
/
"***»
.v
\ •/ %
s*
o
•
r
>
■%
"N
>
/
^^
*
a t
';
■ ■■ >i
<
. -J
O
V
■
Ci
—
10
T
1
r
fllr
f
c1
1
7
fV
if i
i
T
[
>
n
'~-
—
—
._:_ —
J.»
i«
U1
VJ
m
7
"4'"
01
o
f
>
^
»
T-
7
i
2
o
♦«3
o
o
■>
2
d
o
ft
,
-■
—
7—
u.
u.
a
"1 —
u
UJ
.„..
T
o!
i
»-— -«j—
i
i
V
1
1
i
o
Cy
—-
ai\ V Nitia 3S —.
o
t
s s.
^
1
< 0 CL
■
■:>«
:::
>
X.
,-^
■si.
•
O
<*<
>
c c «t
/
::;:
: :
:.
\ •
s
,.*'''
v::
\
r
**»!
0
a
1 N3n « 3c {
VM S /I d
o: >s V
^ <.
/
S
■>
N
^
*^.
'
——
1 \\3 V 31 at
CD
. : .
:
UJ
2
' m <
■ ■: a
u
OQ
; o <
: <
i.i
o
w
—1
ULi
>
a
z
'"iL
....
....
I
„„
—
-„.
.—
—-
...
—-
—
.._..
.: .
*
..
.. :.. .
■
•
•••-r-
... ;._..
.„4-1
1
ji
!
\<
1
1
i
,
' ':' '
- [- -
S-.
j
1
i
—
... j....
- •
;. 4
>
j
/1
j
!
i
:
!....
i
A.
4i
4
f
s i
^^^ i
! \
>!!
i
J
X
"IT"
i
t
l
t
i
!
^
„.,
,,....
....
—
—
—
.'T
>.
i.
./^
^
i
1
' ' '
!
t
1
1
_..-...„
\
4
\
i
'
-^
/
/
**s
\
k
i
I
.-.rV-
•
.-«
t
/
f
.._.
*
)
i
.. 4-.-
_.IJP ....i.o
■"■•[■
•
1
i
11
t
—
1
1
1
ST
--- - <
£
i
J U_JJ_
<
i
I
—1—
i
.._!
" ~T~'
1
—- —
—
4~>
".
I
I
i
I
I
i
|
i
i'
i
; cu
.. .;..©
'1
Si
v
—j—
j
___i__
___ - —-—
'^
V
!
■
1 ^
i
1 low
vws /ijj ONY.
iiii: rfii*
01 3V 3 muo 3s;v
-
. o ....:.o ...: o ..J.JD
cu* . O*
;.
j
;
; .. ■"j-1
r
- 1--
: OQ
w ._ ..
; O wo
—!-<
i
:n
: CQ
: H
' ac
;
<
- :--
---- —
i -1 L3
! UJ
: 2
i C3
... j-gt
\S!M/
i
a
>^
i
1
'
■
—'*e—
.. ..„
H
2
-I
-1
n!
-u,
a
9
..j.o
:
3S:
i
2
■
j
!
i
■ i
•■
-
—
>
a
—
—
■
m
T"'
00
i
—i—
cu
t
i
I
cii
o
t
*&
JL.
!
*
fit
i
1
!
1
j
I
4——-
1
1
|
1
1
i1
!
:
:
•■'
^
• UJ
: u
UJ
2
U
<
■
.aj....
Cflf-
. < U
T <E
:
2
T
J""
-It
2!
—
•■-
. . j.—,
[
_.
,
1
;
:
i- -
■■■<
- <
FT
;
- t
- \ -
/
r
i
i
i
"3?
IX .
jr
1
1
i
i
; O
Lfi
•s. [
/ :
11
L.J....
1 /
;/
I
/ i
... 1./
/
^> S^^-.
!....
;....
,-'
^^
^
.
i
-
. o
I^S
■>
1
^
'
y
■■
... O
•
•
i
i
*
....
*—L
\
7
\
i
j
._: o
i
i
i
/:
/ 5
>
■•^"T
^r... ■X
i
i
\
:\
j \
;
N
, 1/
^
!
1
i
!
4
1
!
—•
.—
»
V
i
:
LNi5
VVi? 880 3?v VNS
|
!.__
:
. o
uj~X
oi""
2i
OI
Oi
■ 2r-
LJi
>!
1
' U •T
i
' ^>
: <
O!
• <
: cn
• -^-
. : a.
Q
: DC
:
*
: _J
■"r<
a:
...;U
t
i *
!
r
.
,
!—-
-—1
r~
—
_o
i
i
.... :....
i
|
1
J. .
1
1
1
T""
r
i
I
—
\
/*
2J
UJ
4si
cT"
P
-i
. ..i.o
. .-• ..-
46
i
,—i—
4
«4.
ii
—i~
I
T
i
rfi
o
1
1 -p
—f—-
L_i.__
si
LT
0
r f-
L. j—
4
«
4... , 9
f—
I. -i. -
S:
NL
J
1""
—i.— J
—
/ml
I
—t— —oi--—
—
'
:
——
r^
1 ^^
1
._J
j
i
j^
3S
aisv
—t—- wf\*
i
...
l/l '
l
—-1
1
-—1
j
i
l
■■-1
- "I
"Z
<
ILt
._- — —
_..
-
i
I
j
>*
LU
_J
.•li4l_
^
2
o!'
2
O
a
it
....
__-
-—
!
i 1-
d
o
< u,
•63
DC
0
;5
- ^5
*
i<rj
: QC
< in
. I—1
1 ^
I <
!
4-Q
■a
i
Hi
1
1
i
.! .
1
\
i
. r—.
!
i
-;-—
i
—1
i
j
i
1
i
-••t -j
;
i
i
!
<
*
■
L
i
•
•
i
:
i
!
1
i
"77
i
4
i
._.iJ
!
t
\
i
|
-j1
1
i
;—
^ •^
..j_. .
-jr
\
-- ,II| *
!
1X
^N ■v.
\
^
^^
^>
\
—.—
i
■T^
I
.__.
——
1
(~
\
/
f
/
\
/
\s
p*
*>
N.
*»■ '
v:
...v
v
s
^
,.
/
: :
^
^
^ •^
\
!
•^' *
:>
\
f-
r
Q
O•
i
i
r
^
\
*»■
_._
'-^
i^-
f
=-=;
'
_c
^_
s
^^
s
. •''
•s'
/
\
\
—
**
"■>.
-— —
/V
',*"'■^.
"oc '
.-
—
^
!
i
i
—i
^
#
i
4
-:—
—
■
-
IT
i
i
F f
i
•i
f
A.
i
.a—
oi
1
1
>'
/ >.
_J
<
H
■
o
oi
—
r
!
T
i
9
CO
cu
cu
CM
\
I
1
I
!
1
1
j
!
j
1
1
1
1
4
i
:
:
j
-j
>
<
LA
49
i ""
58—
l
"4"
-r
i
o
i
i
Nil
,
OI
j.-- .
i
I
1
1
..-i-_
)
.-
I
— ~"!—
/ *"
H
u.
Hi
1
,.1.
d
.._j.fi
- ..j.—
T"""
i
i
IN 3: > * i3 d ' M i
ayo 3SV VJ ^s VI d dNv nna ^
_„!Q!; >V;3
i
•
:
*
I
!
CHAF T ]¥ E.
i
i
!
M EAN DAIL visi RUM AMD PLASMA ASCORBIC ACID VALUES
A< :iD INTAKt
01 : FOIJ R :R( VS clw KhJoWN L^VI EL$ i )F ASCORBIC
1
!
!
j
i
•
j
,
;
.60
1.1
,
-
- -: •'
.40
CO
■ <
■—:&■
i
:4
i-J
~T&"
'.
:UJ
i
i
•—r— -
XAO
Kao
.a
Z
00
cc;...
1
■ (/I LU
/ •
7
i
>r
!
[
|.?0
jjjr—-
-T^
/\
--:----
h.
/—
7
! x
'.. i
<
>
•
1
1
j——
'
[
J
1
l
1
j
|
*
!
i
-— f--
^
v-i
r"
/f
i
"■
. —^
^'
—:....
*^j
u_. —--) —
i
nr^r
"^N
--U —
',
i
^
—--1—
i
;
j
!
U—
: rf»
: s
t
i
^r--
—r ■-
1
■ -- j
—f — —j....
( ^0
<:
— —i—
i
1
i
"Is
i
j
i
—- -
1
". / \ ;
\
----'—- ... 4- —■ -—:--- ——:—-
.. .:
:
\^_
/
---
!
— i--V
/
• !
:
.........
" "■""
!
i
-—i—
uo
o
:
-i—-
<
6
\
j
[
'
!
■
-g
!
' P"
r*. r- r-t
!
■ -~:—-
—- r
■
_-_L-_^
PMSMA
i
^--2 QQ.jyiC ..SUP >.LEM£ \ir_
\f
i
4
!
. joo Mfr.T >XAL
^v/S
I
:
'
j
SOM.&...riiT4L. ——^
i
-<»»:» II
ID
12 ; -l|4| 16-4 IB -j 20^
22-4 24
26■ : 5fr
;
30
•
_i_..
!
;
i
iw i
..I-
_
:
©
51
CHAFPBR V
.
INTBRPHmTIQK OF STATISTICAL ANALYSIS OP THE DATA1
Th® analysis of variance was used to find out whether
or not there were significant differences in the ascorbic
acid content of th© blood serum from one teat period to anotherj naisely, saturation period, the period wh®n th© subjects received the recommended allowance of the National
Research Council and the period when they received 10 mg*
less than the National Research Council*© ree©m*a©ndation*
For this analysis (method of analysis in Table IT)
th© data for th© last 5 days out of each 10»day period
were used excluding the data obtained during the first 5
days of each period trahen the subjects were becoming adjust*
ed to a new level of ascorbic acid intake.
recorded In T&bl© II*
The results are
In this study,, the mean of the Satu-
ration period is significantly higher than the means of the
period of 100 mg» ascorbic acid intake and th© period of
90 mg» daily ascorbic acid intake*
Eowevcr,, the difference
between the means of the period of 100 sag* dally intake and
of the 90 aig. intak© is insignificant*
This indicates that
a daily intake of 90 mg* of ascorbic acid was as
1 The -writer is indebted to Dr. J. C* H, hi for assistance
in the statistical interpretation of th© data obtained
In this study*
52
satisfactory as an intake of 100 lasg, aaoorbic acid.
By this sam© jaethod of statistical analysis, it was
found that individual differences In th© response of th©
variows subJoets were not statistically signifleant»
§3
TABLE IV
Statistical aaalysis of th© data to detormin© th©
signifioaao© of th© diff©r©ne©s in th© B©rma
total asoorM® acid imlaes of three ©3iperim©a.-teil periods
Sublet
f.C.
Sema
A.A,
8.1.
Serum
A .A.
Mg. %
Bat©'
'm'."y
(1)
Satura**
tion
Period 2/6
2/7
g/8
2/9
2/10
1.04
1.38
1.20
1.27
1.55
1,00
1.28
1.28
2/16
2/17
2/18
2/19
2/20
(5)
90 %*
Period 2/26
2/27
2/28
2/29
3/1
xr.p. ■
Serum
A.A» •
D*H*
Serum
1
A*A«
Hg. %
" ' i^r w ^
i*4i
1*35
1.70
1.65
1.46
1.50
1.50
1.55
1.62
1.35
1*50-
1.20
1.83
1.35
1*21
1.11*
I.05
1.15
1.03
1,20
1.08
0,98
1.15
1.41
1*29
1.16
0,90
1.20'
1.08
1.15
1.08
1.26
1.29
1.29
1.22
1.19
1.35
1.17
1.18
1.18
1.07
0.87
1.25
1.2?
1.29
1.16
1.28
1,21
1.19
1.23
(1)
6,44
J
6*37
5.51
5.95
7.81
5.99
5,80
7.43
5.41
28.05
6*13
6.25
6*08
Si4.08
18.82
17.83
19.60
18.92
75.17
(2)
I.S.C.
Period
2
(3)
prgiaal
fotal
XM
1
1*23
I
1.4025
1.1520
1.2040
54
fABLE I? (oont.)
T
persoa
Period
Group
Error
Total
Correotion
Htli*. 2277
1897^905
li76.ij.081
9li#2819
9l*.87U5
95.2816
95»9^1
5650.5289
95,9991
9U.1755
Analysis of Varianoe
Variation
Person
Period
ss
d.f.
0.106U
0.6990
3
2
Error
Total
Varianoe
0.035^7
0.31*95
f
.7077 Wot signifioaat
6.9733 Signifioant at
3.353
6
0.^007
0.05012
i*8
0.7175
1.8236
0.0lij95
lateraotioa
0*10614.
0.6990
1.1061
0.7175
I.8236
5 % level
Sigrdfioaat at
5 % lev©!
S.E. of th© differexio© botwesn any two period ffie^aa * /Ss
a. /^m^m^r
»
VK^SK-
* 0.07^795
fh© loast sigaifiooiat diff©r©Boo (at ^) betroen aay two period means
is*
• to.05(O*O7O8O)
(1)^(2)
(1) • (3)
(2) * (3)
a
(2,l4U7)(0.O708O)
a
0*1733
a 1,^025 • 1,1520 s .2505
* 1#U025 * 1.20U0 « .1985
s 1,1520 * 1,20140 * .0520
The mean of period (1) is signifioantly (at ^) higher them th© means
of psriode (2) and (3)» ku^ ^^® diff©r@no© between tho aea&s of
psriods (2) aad (3) is insigaiflcant*
Th@re is aa interaetioa between p©ri0d aad person.
55
Statistioal analysis of th© data to detesTnia© th©
signifloane© of th© differenoes batsjoen
serum (t@tal) aad plasma (r©&uee&) asoorbio aeld
Subjeot
f.O.
Serum
Plasma
A,A.
(1)
Satura*
tton
Period 2/6
(2)
I.E.C*
Period
Serum
Plasm
AoA.
.37
.36
.39
.56
.29
.6^
.66
•lij.
.33
J48
.29
.12
32
30
23
.57
2/7
2/®
.51
.06
2/9
2/10
.2§
2/16
2/17
2/18
2/19
2/20
Serum
Plasaa
.21*
.i}6
.34
.37
.55
.54
.32
.P.R.
Soruia PlasEia
A.A.
ig«
4o
68
26
32
.18
.56
.38
.45
.37
(5)
90 1%.
Period
(1)
(2)
(3)
Marginal
fotal
2/26
2/27
2/28
2/29
3/1
.53
.36
.37
.68
.51
.itO
.47
.53
.37
1.47
1.65
2,07
1.97
2.13
2.56
5.19
6.66
,18
.54
.54
.41
.45
.47
.38
.36
.64
.55
§s
If (oont.)
x2
a«
Porsoa
Period
Group
SSrror
Total
Oorreotioa
150.1757
200.1565
50*6077
Divisor
15
20
10*9989
595,8^81
5
Divisor
10.0117
10.0078
10.1215
1
60
10.9989
9*9308
ss
,0809
.0770
.1907
*877k
1..0681
Amlysie of Varianoe
Variation
Serum-Flasma
Signifioant
differene®
lo eigaifieaat
difforene©
So eigaifioaat
P©r80B
Period
differ ®&o.e
Ho si^aifioant
<|iff©r©no©
Iat@raotioa
Error
total
fhis analysis shows that ©©ma is higher thaa plasma^ and this
difference (Serum ** plasma) does aot vary from person to person or
frcro poriod to period*.
The 95 % eoafideao© i&tsrval of population moan of the difforeaoe
betcireen seruan aad plasm is8
'■'la1'
0.1407
s o»l«07
s
£
s
O.i+O?
0.ii07
0,1*07
0<373
or 0.37 *
d
>.008A //s
*****
1/60
60
2.008.
•
"IS
2,008(0.017)
0,051*
popnilatioa differeae©
O.iM
67
CHAPTER ¥1
SUMAKZ Aim COSCLUSIQHS
Th® micro determination of th© total ascorbic acid
content ©f blood serum of adolescents is described*
Serum total ascorbic acid values were determined daily
in four adolescent boys {18-year old) for three t©n*day
periods, during ushich time the intake of ascorbic acid was
controlled.
The mean serum total ascorbic acid content during the
saturation period was 1.40 mg. per cent and ranged from
1.27 to 1.56 mg. per cent.
In the period when the dally
intak© of ascorbic acid was 100 mg, the mean aerum ascorbic
acid value was 1*15 sag. per cent and ranged from 1.0S to
1.88 rag. per cent.
The mean serum ascorbic acid content
when the subjects received 90 mg. daily was 1.20 mg. per
cent and ranged from 1*1© to 1.25 mg* per cent*
There was
no si^iificant difference between the last two means.
The
means for the saturation period were slgaificantly higher
than the means of the last two periods.
It can be concluded from this investigation that a
90 mg. daily intake of ascorbic acid for adolescent boys
is as good as the 100 mg. daily intake which is the
aecojnmended Allowance of the national He search Council.
Th© mean difference between serum total ascorbic acid
and plasma reduced ascorbic acid content was 0.41 mg. per
58
cent.
Prom the data obtained one could i>r©dlct statisti-
cally that the difference between the serum total ascorbic
acid content and the plasma reduced ascorbic acid content
would vary from 0.37 to 0.44 ag. per cent in 95 per cent of
the cages.
As tested by analysis of variance these dif-
ferences xvere independent of level of intake of ascorbic
acid or individual variation among subjects0
59
1,
Abt, A» P., Farmer, C. J. and Epstein, 1. 1» Hormal
0©^itamic (ascorbic} acid detarminatiofts in blood
plasma gfcnd tiieir relationship to capillary
rstaiatanceu J. F©diat* 8il-19,. 1936*
2«
B©ls©r, W. B#, Hauek, H. ffi. and storriek, C. A. A
study of the ascorbic acid intake required to
maintain tissue saturation %n normal adults,
J. Nutrition 17t5i3-526, 1939.
3.
Beasey, 0. A.
sourcea,
4.
Besseyj, 0<. A. A method for the determination of small
quantities of ascorbic acid and dehydroascorbic
acid in turbid and colored solutions in the
presence ©f other reducing substances. J, Biol*
Cheau 126}771-784, 1938b.
5.
Beas®y> 0. A. and King, G* 0. fhe distribution of
vitaaiin C in plant and aniaal tissues, and its
determination. J. Biol. Chem. 103t687-698, 1933*
Vitamin C, Methods of assay and dietary
^* Am. Med. Assoc. 11121290-1298^ 1938a,
., 0. A»» Lowry, 0. H. and Brock, M. J. fhe
quantitativ© detearanlnatlon of ascorbic acid in
Small amounts of whit© blood cells end platelets.
3* Biol. Chem* 168*197-205, 1947.
7.
Butler, A. M., Oushman, M. and
determination of ascorbic
Its constituents by means
macro** and micromethods.
461, 1943*
lacLachlan, B. A. fhe
acid in whole blood and
of laethylene blue?
j, Biol* Gh&m, 130$453-
8.
Chaney, I. S. and Ahibom, M. nutrition. 3rd Bd«
Houghton llfflin Company, 1943i Section 4.
©i
Chu, Fi T* and Chow, B. F* Correlation between vitamin
0 content and complement tlter of human blood
plasma. Pro©. Soc. Ixp. Biol. Med, 38j©79*682,
1938;
10.
Dann, M* The influence of diet on th® ascorbic acid
requirement of premature infants. <J. Clin* Invest.
81*139-144* 1942.
60
11.
Feiraer, Co ^r. and AbtP Ao p. Deterraination of reduced
aacorbic acid in small amounts of blood. Proc.
So©. Exp* Blol. M«dv 34sl4S*l§0, 1936,
12.
Faullaier, $<> M. and Taylor, F. H. %* Observations on
the r©nal threshold for ascorbic aeid in man.
J. Clin. Invest. 17j69-75, 1938*
13.
Gothlin, G. F. Human daily requirements of dietary
ascorbic acid. Hatur© 134s569-570j 1034.
14.
Qreenberg, %, D., Rinehart^ J. F. and Baatak, H, M.
Studies on reduced ascorbic acid content of the
blood plasma* Proc- Soc. Bxp* Biol. Med*
35:135-139j - 1936*
15.
©reenwaldj G. K. and Hard®, E* Vitamin G and diphtheria toxin. Proc. iBoe* Ixp* Biol. led* 3S:11S71160, 1935.
16.
Harden, A. and Zilva, S. S. $he antiscorbutic factor
in lemon juice. Bioehem* J* 12t259-269, 1918.
17.
Hess, A. F. and Benjamen, H* R. Urinary excretion of
vitamin C. Proc* Soc. ISxp. Biol. led.
31:855-860, 1934.
18.
Hess,. A. F« Scurvy, past and present.
$4 B* Lippincott Company, 1980.
19.
Holmes, P. E*, Cullen, G* 1. and nelson* W. S. tevels
of ascorbic aeid in the blood plasma of apparently
healthy children. 3. Fedlat* 18:300-309, 1941.
20.
Hoist, A. and Frollch, T. On the etiology of scurvy.
J. Syg. 7:634-671, 1007.
21.
Johnson, S. W. and Zilva, S. S» The urinary excretion
of ascorbic and dehydroa^corbic acids in man,,
Bioehem.* Jf, 28:1303-1408^ 1034.
22.
Hine, A* B. and Sheart, M. S* Variation in t3ae
ascorbic acid requirements for ssturation of nine
normal young women. J. Nutrition 28:413-419,
1944._
23.
King, C. G. Chemical nature of vitamin C*
75$337-353, 1032.
Killadelphia,
Science
61
24.
King, C. G. and Menten, M. L. The influence of vitamin C level upon resistance to diphtheria toxin.
J* Nutrition 10:129-140, 1935.
25.
Lewis, J. S., Storvick, C. A. and Hauck, H. M. Renal
threshold for ascorbic acid in twelve normal
adults. J. Nutrition 258185-196, 1943.
26.
Levine, S. Z., Marples, E. and Gordon, H. H. A defect
in the metabolism of aromatic amino acids in premature Infantat the role of vitamin C.
Science 90:620-621, 1939.
27.
Levine, S. Z., Marples, 1. and Gordon, H. H. A defect
In the metabolism of tyrosine and phenylalanine in
premature infanta I. Identification and assay of
intermediary products. J. Olin. Invest. 20:199207, 1941.
28.
Loeffler, A. J. and Ponting, J. S. Ascorbic acid.
Rapid determination in freah, frozen, or dehydra*
ted fruits and vegetables. Ind. Eng. Chess.,
Anal. Ed. 14:846, 1942.
29.
Lowry, 0. H. and Bessey, 0. A. The adaptation of th®
Beckman spectrophotorn©ter to measurements on
minute Quantities of biological materials'. J.
Biol. Chem. 163:633-639, 1946.
30.
Lowry, 0. H. and Bessey, 0. A., Brock, M. J. and
Lopez, J. A. The Interrelationship of dietary,
serum, white blood cell, and total body ascorbic
acid. J. Biol. Chem. 166:111-119, 1946.
31.
Lowry, 0. H., Lopez, J* A. and Bessey, 0. A. The
determination of ascorbic acid in small amounts
of blood serum. J. Biol. Chem. 160:609-615,
1945.
32.
Mitchell, P. H. A textbook of biochemistry, new York
and London, McGranr-Hill Book Company, Inc. 1946
152 p.
33.
lusulln, R. R. and King, C. G. Metaphosphoric acid In
the extraction and titration of vitamin C. J.
Biol. Chem. 116:409-413, 1936.
32
34.
lational Research Gouneil. Committee on food and
nutrition. KecoHunended dietary allowances. Hevised reprint and circular series. Ho. 122, 2 p.ipi^.
35.
Ralll, I* P., Friedman, S. J* and Sherry, S. The
vitamin C requirement of man. Estimated after
prolonged studies of the plasma concentration and
daily excretion of vitamin C in 3 adults on controlled diets. J. Clin. Invest. 18*705-714,
1939.
36.
Eoe, J. H. and Kuether, C. A. fhe determination of
ascorbic acid in whole blood and urine through
the 2,4-dinitro5h©nylhydrazin© derivative of
dehydroascorble acid. J. Biol. Chem. 147j399407, 1943.
37.
losenberg, H« I. Chemistry and physiology of the
vitamins. Hew York, Xnterscience Publishers, Inc.,
1945. 289-337 pp.
38.
Sargent, F* A study of the normal distribution of
ascorbic acid between the red cells and plasma of
human blood. J* Biol. Ghem. 171t471-476, 1947,
39*
Sherman, H* C. and Smith, S. L. The vitamina let?
"STork, One lad is on Avenue, Book Department, The
Chemical Catalog Company, Inc. 1922. 142-146 pp.
40*
Sherman, K. C. Chemistry of foods and nutrition.
7th Ed. lew York, The Macmillan Company 1946.
321-347 pp.
41*
Sigal, A. and King, C. G. The relationship of vitamin
C to glucose tolerance in the guinea pigs. J.
Biol. Chem. 116:489-492, 1936.
42.
Smith, S. L. Human requirements of vitamin C.
i. Assoc. 111*1753-1764, 1938.
43.
Storvlck, C. A,, Fincke, M. X,., Quinn, J. P. and
Pavey, B. L. A study of ascorbic acid metabolism
of adolescent children. J. Futrition 33:529-539,
1947.
J. Am.
63
44.
Storvlok, G. A, and Hauck, H<, M. Effect of controlled
ascorbic acid ingostion upon urinary excretion and
plasma concentration, of ascorbic acid in normal
adults. J. Nutrition 23:111-123, 1942.
45.
Sa@nt-©yorgyi, A. and Haworth, W* H. 'Hexwronic acid'
(Ascorbic acid) as th© antiscorbutic factor*
STatur® 131 j24, 1933.
46.
The Vitamins. Chioago* American Medical Assoeiation
1939. 354 p.
47.
Wauga, W. A. and King, C. ©. Isolation and Identification of vitamin C. J. Biol. Chem. ©7*325-331,
1932.