CHAMES IH CGKCEITRATKMS OP EXBOPLAVm XS HOISM
BLOOD PRACTIOIS POLLOWIHG TEST DOSIS OF THE VITMISI
by
SOO JAB aOOM
A THESIS
submitted to
OREGQM STATE COLLEGE
In partial fulfillmeat of
the requirements for th©
degree of
MASTER OP SCIBJCE
June 1960
APPBOVED:
Associate Professor of Foods and Nutrition
In Charge of Major
<fas3Jfait.m,m uiStggapsoT'M'tvwi
Head of Department of Poods and lutrition
Chairman or School Graduate Committee
Dean of Graduate School
Date thesis is presented
Typed by Vema Anglemier
July 29« 1959
ACM OKLEMEBWTS
The mthw wishes to express her sincere appreciation
to Dr* Betty E» Hawthorne for her careful direction of tMs
research* continuous encouragement, her generous participation in it, and her willing assistance in the preparation of this laanuscript.
Grateful thanks are extended to Br* Clara A. Storvlek
and Dr. Margaret L* Fincke for their kind advice and
interest during the progress of t&e study*
The mthor is greatly indebted also to those tsho
participated so cheerfully as experimental subjects and
laboratory workers for their cooperation and interest in
this study.
TABLE OF COIf BffTS
Pag©
DTTRODUCTICB •♦*.»-.....
1
RBVlECf (F: LITERAfHE
S
mp!Rira?m
12
STUDY
Plan of the Study
IS
Description of tho Subjects and Diets
Analytical Method
....
..*•'...
Principle of Method
12
14
'
14
Equipment ................ o
15
Keag^ts
1?
Method of Analysis of Free plus Rffl and
Total Riboflavin in Blood Fractions ....
20
Collection and isolation of blood
fractions ...............
20
Method of analysis of serum
22
......
Method of analysis of red cells
. . . .
23
...
24
Measurement of phosphorus in white cells
25
The preparation of control samples for
blood fractions . • . *
27
Method of analysis of white cells
TAB£1 OP CCMTMTS
(continued)
Page
Calculation of Results
....,.«...
28
• . . .
29
• • . « • . .
29
. « . . • .
50
Calculation for actual free plus WM and
FAD riboflavin in blood fractions ♦ . .
SI
Eeprodueibilitj of Method in This
Investigation .<> ......*..... ,
31
Calculation for serum
Calculation for red cells
Calculation for Ait© cells
RBSULfS MD D1SCUSSKM
,•*».*».••,«««
S3
Changes in Concentrations of Hiboflavin in .
Blood Fractions After th© feat Dog© of 2 nsg
of Rifoof lavin # ... ..,»<» ...,»♦ ,
55
Tina Concentrations of Hiboflavin.in Serum «
35
The Concentrations of RiboflavSn in Red
Cells ...................
39
Th© Concentrations of Hiboflavin'in White
Cells ....... . o ......... .
40
Discussion
42
........ o ...,>.. .
Comparison of fuo Levels of Test Sose
Comparison of Duplicate Tests
STOMRY
BIBLIOGRAPHY
, • . , ,
« . • .
44
........
47
50
52
UBf m $ms8
?abld
Pago
folMttog a f rag f@st i^s© @f Si^ofIdtrte •
3
&e&<»fflptlQKk ©f S^fej^dts
......
4
Gonoontratioos of ffotol iibsflavis fiotesnatoed
e
.. .
10
%&
f©il©^nf a f s® f0st Doss ©f Hlb^l&vto »
34
FollooSng Too Xid^old «f ®©st B®s© f<*P III
46
©©©©s of Bife©flQVia for SffiS
4®
«•♦,»•♦
LIST OP FIGURES
Figur©
1
2
3
4
iP-age
Total Riboflavln Concentrations in Blood
Fractions Following a 2 mg Test Dose
of Biboflavin «..„...
Free plus BM Riboflavln Concentrations
in Blood Fractions Polloi'jing a 2 sng
Tost Dose of liboflavln » ♦
36
*
37
FM) Hiboflavin Concentrations in Blood
Fractions Following a 2 mg Test Dos©
of tiboflavin
38
Total Riboflavln Concentrations in Blood
Fractions of BIH Following Test Doses
of Riboflavln ..............
4©
CHMSSS ZH CCEfOEWTRASPICHS OF EIBOPLA?IH IN EOSSAN
BLOOD FRACE[OIS FOtLOWMG TEST DOSBS OF THE VITMIH
OTROMGTM
The importance of riboflavin in the nutrition of
animals and of man has be ©a investigated by a number of
biocheaists, physiologists, and nutritionists in the last
quarter century since the discovery of riboflavin.
In his
recent review Bro-Rasmussen (6, p. 1) states, "Riboflavin
acts as a coens^fme *.<,. both of dehydrog©nases which act
specifically on aaiino acids, and of enzymes which act in
intermediary metabolism on products that arise from the
metabolism of carbohydrates and fats."
Most tissues show
a remarkable constancy in riboflavin content.
lh©n the
riboflavin content of organs is examined in relation to
the intake of the viteatin, relatively small variations
occur between maximum and minimum values (6, p. 1*23).
It has been reported also that there are no organs tshcre
riboflavin is stored in the animal body (11, p. 1157} 15,
p. 514-516).
Blood is a tissue that circulates in what is virtually
a closed system of vessels.
This circulating tissue per-
forms the important functions of transporting nutrients
and oxygen to all the cells and removing th© waste products
of their metabolism.
Blood consists of solid elements.
the red and ?Mt© cells and the platelets, suspended in a
liquid aedium, the plasma.
In early investigations of riboflavin in whole blood
using Merobiologieal methods, either no variation could
be demonstrated, Axelrod et al. (1, p. 146-149), or the
changes in riboflavin concentrations found could not b©
related to riboflavin intake. Strong ©t al. (22, p. 563S7S)*
In 1948 Burch et ^. (7, p. 457-470) introduced a
more sensitive cheaieal aierosnethod which made possible
the determination of the riboflavin content in blood fractions as well as in whole blood.
Since 1948, interest in
the riboflavin content of blood fractions has attracted
several investigators, e.g., Burch et al, (7, p. 457-470),
Bessey et al. (4, p, 367-385), Suvamakich jgt al* (23, p.
105-118), and Wa et al* (27, p* 231-240).
The purpose of this investigation ms to determine
the sifflultaneous changes in cmc cat rations of riboflavin,
as free plus flavin mononucleotide, flavin adenin©
dinucleoUde and total riboflavin, in serum, red cell, ©nd
white cell-platelet fractioas of blood at intervals for
three hours after a 2 sag test dose of the vitaudn was
administered to subjects,
fwelve subjects, nine women and
three men, were studied.
This investigation was part of a
research project on the relationship of certain vitamins
to cholesterol in human blood fractions.
REVIEW OF MTBRKFURE
Riboflavin, a water-soluble, yellou-green fluorescent
pigment, occurs in -Hiree forro in animal said plant tissues!
as free riboflavin, in flavin mononucleotide (MB),
and in flavin adenine dinucleotide (FAD)*
it has been
reported by Wagner-Jauregg in 1954 (24, p. S01-S09) that
only the free form is found in the retina of the eye, in
whey and in urine,
in most tissues, riboflavin is present
as POT and FAD as nell as in the free form.
The FAD form
is tsrildely distributed in animal tissues and in microorganisms.
Bessey, Ikmry, and Love (S, p. 755-76©) found
that 70 to 90 per cent of the total riboflavin in all
tissues is present in the form of the dinucleotide.
Riboflavin has been shown to be a constituent of a
number of enzyme systems associated with tbe intermediary
metabolism of carbohydrates, proteins and fats.
The first
enzyme found to contain riboflavin was the ye 11 OH enzyme
of Warburg and Christian, recognized in 1952-1955, which
assists in the oxidation of hexose*phosphoric acid (12,
p. 3S4-357).
Riboflavin is also a constituent of D~amlno
acid oxidase* L-amino acid oxidase, hydroxy acid oxidas®,
xanthine oxidase, succinic acid dehydrogenase, aldehyde
oxidase, reduced triphosphopyridine nucleotide-nitrat©
reductase, reduced triphosphopyridine nucleotlde-cytochrom©
4
c reductase, gljcine oxidas©9 diaphoraa©, acetyl coeng^m©
A dehydrogenasos, glycolic aoid oxidase* and fiamaric
dehydrogenas© (10, p. 334-357)♦
All of these enzymes
fmetion in oxidation-reduetion reactions in th© cells of
th© body»
is FAD.
Th© prosthetic group of most of these enzymes
Bartlett reported that th© riboflavin in FAD re-
mains at a more constant level than the free riboflavin
in tissues (2, p* 15S-168). She stated, "FAD reflects th©
more constant internal metabolic demands and therefor©
chaKiges in th© level of th© PAD in tissues may b© indicative of th© nutritional status of th© body as whole" (2, '
p. 166).
Riboflavin is found in th© blood in all three forms:
as free, EM, and PAD riboflavin.
th© serum and th© cells,
Shis also is true for
Kleim and Kohn
in 1040 (16,
p. 177-189) first reported that human red blood cells
could s^fnthesiz© PAD from fre© riboflavin both in vitro
^^ i£L lil2»
^e si©ch©nisii of the transformation of fr©©
riboflavin to FAD in blood cells in not knotfn, however
(12, p. 334-357).
Studies of riboflavin concentrations in whol© blood
and in blood fractions hav© been reported by a number of
investigators who used micro biological and chemical
methods.
B@vi©w©d data ard summarised in Tabl© 1*
For random blood samples from adult subjects on
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unrestricted diets, total riboflavin eoneaatrations In
whole blood which have been reported renge from 12.2 to
S4»0 meg per 100 ml (1, p. 146-149S 5, p. 379-385; 7, p.
457-470! 19, p. 206-211? 22, p. 363-372).
fotal ribo-
flavin concentrations in aerum for random blood samples
for subjects on unrestricted diets rmiged from 2.6 to 3.7
meg per 100 ml of ttiich 0.3 to 1.3 meg per 100 ml was in
the form of free plus FSM riboflavln (7, p. 457-470).
For
these same subjects concentrations of total riboflavin of
the red cells ranged from 18.0 to 26.2 meg per 100 ral and
of the vjMte cells from 227 to 293 meg per 100 ga (ibid.).
Fasting blood samples of subjects on unrestricted
diets have also been analysed for riboflavin.
Wu (25)
reported concentrations of total riboflavin in serum
ranging from 1.6 to 5.7 meg per 100 ml for 29 subjects on
unrestricted diets.
Samvik (17) determined concentra-
tions of total riboflavin in fasting whole blood, red cells,
white cells and serum for 59 subjects.
Concentrations
ranged from 4.3 to 14.6 meg per 100 ml of whole blood,
from 7.1 to 25.7 aeg per 100 ml of red cells, from 162 to
292 meg per 100 gm of ifhite cells and from 1.8 t© 5.5 meg
per 100 ml of serum.
In addition, concentrations of riboflavin in various
fractions of fasting blood have been determined for subjects on known riboflavin intakes for appro3d.aately 30 d&j
periods.
Eiboflavin concentrations of fasting serum
samples from seven subjects were detorained In this lab*oratory by Wu» Warren and Storvick (27, p. 2S1-240).
The
subjects received a total of 1.2 mg of riboflavin daily*
Concentrations ranged from 1.7 to 6.3 and from 0.2 to 4.2
meg per 100 ml of serum for total and free plus WM riboflavin, respectively.
Concentrations of total riboflavin
in whole blood, red cells ©nd tshite cells as well as in
serum for an additional seven subjects were determined in
this laboratory by Edwards (9) and Irgens-BH^ller (14).
Total riboflavin concentrations ranged from 6.5 to 11.2
meg per 100 ml of whole bloods from 7.5 to 16.8 meg per
100 ml of red cells,, from 199 to 293 meg per 100 gm of
white cells,, end from 2.0 to 4.5 meg per 100 ml of serum.
Concentrations of riboflavin In th© serum ©nd in th©
cells have been observed to be affected by the consumption
of different levels of riboflavin over long periods.
Suvamakich et al. (23, p. 105-118) found that serum rib©*
flavin concentrations diminished with decreasing rlbo*
flavin intake.
The change uas greater in the free plus
MM than in the FAJ3 riboflavin.
Bessey et al. (4» p.
367-383) reported that for a group of six subjects on
riboflavin intakes of 2.55 to 3.55 mg per day for 16
months, the mean total riboflavin concentrations were
'22.3.meg per 100 al of red cells, 212,meg per 100 gm of
3
white cells sad 3.03 meg per 100 ml of serums 0.71 meg per
100 ml being present as free plus WH riboflavin.
On the
other hand* another group of twelve subjects on 0.55 ag of
riboflavin for 16 months had mean total riboflavin concentrations of only 11.7 meg per 100 ml of red cells, 192
meg per 100 g?a of vhit© cells, and 2.4 meg per 100 ml of
serum, 0.3 meg per 100 ml being present as free plus WM
riboflavin.
Comparing the data from the two groups*
Bessey and co-trorkers concluded that the riboflavin content of the red cells was the most sensitive and convenient
index of riboflavin intake.
Only two published references reporting concentraticaae
of riboflavin in a blood fraction following a test dose of
the vitamin were found.
Both were studies of changes in
serum riboflavin concentrations only.
In 1948, Burch ejb
al, (7, p. 457-470} reported graphically changes in concentrations of total riboflavin in serum of three subjects
who were given 2 mg of riboflavin as a test dose.
The
concentrations of riboflavin were determined at fasting
and one-half, one, two, three, four, end five hours after
the test dose.
The subjects were previously restricted to
riboflavin intakes of 0.4 mg per day for four days.
These
researchers found that the greatest increase in serum
total riboflavin occurred at one-half hour and the concentrations ©f riboflavin returned to approximately initial
levels in from two to five hours.
Ifu, Warren, and Storvick
in 1963 (27, p* 231-240) experimented similarly with four
subjecta using a 2 mg riboflavin test dose.
Th&j observed
that both the free plus FMS and total riboflavin of the
serum reached a peak in one-half to one hour after the in*
gestlcn of the test dose*
Thereafter, the free plus Ffflf
riboflavin returned to the fasting level by the end of the
second hour but the total riboflavin decreased mor© ssloroly
and did not reach the fasting level until the fifth hour.
Changes in concentrations of riboflavin in whit®
cells and red cells as vjrell as in serum tsrer© investigated
in preliainary studies on five subjects in this laboratory
during 1953*1954 by Sauvlk "(20) and Wu (26).
These unpub-
lished data for six tests (two tests for on© of the subjects) are suanaarised in fable -2,
fhe highest concentra*
tions of both total and free plus WIM riboflavin in serum
occurred one-half hour after the test dose and the concentrations of total and free plus PEM- riboflavin decreased
toward initial levels in from too 'to four hours.
Concen*
trations of FAD in serum showed only slight changes after
the test dose.
Changes in concentrations of riboflavin
in red and t&it© cells uer© leas consistent than changes
in the serum., ?ery little change in both total and FAD
concentrations in red cells was observed in four of the
six tests.
In- too tests FAD and total riboflavin
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concentrations Increased.
Only small changes in total
riboflavin concentrations of white cells were observed.
Total riboflavin concentrations increased from fasting
values in two tests, decreased in two tests, and in two
tests there was essentially no change.
12
Plan of the Study
The concentrations of riboflavin in blood fractions
of twelve subjects were determined following a test dose
of tke vitmoin.
Blood samples were obtained at fasting
and one~half, one, two, and three hours after 2 mg of
riboflavin vms given with a teat meal of salted crackers
and black coffee.
Each sample of blood vms separated Into fractioas'sserum, red cells, and whit© eell<*platelets.
Serum and red
cell samples wr® malyzed for free plus RMS FAD and
total ribof3avin»
White cell sampler were analyzed for
total riboflavin for all subjects and for five subjects
white cell samples were analyzed for free plus MM, PA©
and total riboflavin.
Tuo additional tests were included in the study.
The
response of one subject was measured following a 5 mg as
well as a 2 mg test dose of riboflavin.
itoother subject
was given a second 2 mg test dose on an additional day.
Description of the Subjects and Diets
Data for age, height, weight and sex of th© twelve
subjects are shown in fable 3.
Each subject was asked to
13
fabl© 3.
Subject
©©scrjlption of Subjects
Sex
Age
Height
Weight
Years
Inches
Lbs*
Younger vjommt
BBS
F
38
68^
US
30?
F
32
60
110
SSAE ,
p
44
®4|
140
SJM
P
25
61
110
HI
F
38
63
134
MB
F
31
65|
133
^F
F
58
65
144
BAG
F
66
66
150
65
F
66
65
136
®m
M
44
6©|
138
EBV
'M
40
69
145
<M
1
29
68
180
OM®!* wome&g
leas
:
14
com© to the laboratory vdthout breakfast and the fasting
blood sample was obtained*
Immediately, each subject was
given a test dose containing 2 rag of riboflavin.
A'2 rag
test dose was used as this ©mount is nutritionally sound
relative to the recosamended daily allowance of the
National Research Council (18, p. 18)*
In addition to
this test dose th© subjects were given 30 gra of salted
eraekers and black coffee ad libitum*
The test dose of 2 mg of riboflavin was prepared aa
follows?
20 mg of riboflavin (WB crystalline) was
weighed into a 500 ml volumetric flask, dissolved and
diluted up to volume with redistilled water ©nd thoroughly Mixed*
Fifty ml of this solution contained 2 mg
of riboflavin.
Analytical Method
Principle of Method
The jBicromethod of Burch, Bessey,' and iowry (7, p.
457-470), as modified -in this laboratory by Edwards (9)
and Irgens-M^ller (14), was used for th© deterraiaation of
free plus FM and total riboflavin in the blood fractions.
The method for analysis of each fraction
involves th©
extraction from the sample of riboflavin and riboflavinoontaining compounds with triehloroacetic acid and the
comparison of the fluorescence of riboflavin to the
is
fluorescence of a suitable cone mt? at ion of a fluor©ac©in
standard.
In order to ealctalat© th© amo\mt ©f riboflavin
in the samples, the use of an int©Kial standard ia employed.
Sine© most biological mat^ials do not yield
extracts xTfhich Contain riboflavin as th© only fluoresc^sit
material^ a blank m©asw©ii©nt for each sample, in 'rahich
riboflavin is r©duc©d to the Imaiflavin with sodium hydrosulfite, is required.
It is assimied that only riboflavia
is reduced and that no fluorescent materials ar© produced
in the redaction (IS, p. S61-S7S).
Simple reagent blanks
do not suffice.
Sine© riboflavin occurs in both fre© and combined
forms, as FW and as FAP, In serua and in cells-, both fr©e
plus F2M and total "riboflavin ar© d©terrained.
fh@ free .
plus MM riboflavin in © s©mple is measured by analysing
the ©xtract directly.
For total riboflavin deteraioations*
the ©3£tract is incubat©d for 20 hours at 37©0 to hydro lyze
FAB to fre© riboflarto prior to analysis.
Since riboflavin is quite sensitive to light* most
of the ©xperlmsntal worfe is best carried out in a dark
room ©quipped with red lamps.
I3hen necessary, saspleg
may be- prot©et©d from light by covering with a black
cloth.
16
Equipiaent
1.
Bard-Park©r blad© lo. 11.
2.
Tubes? 6 x 50 ma.) and pyres tubes of fee following capacities:
10 x 7S raa and 8 ml.
3.
Calibrated pyrex tubes for optical us®, 10 x 70
4.
Small tubes for collection of blood.
S#
Constriction pipettes of the following eapacitios:
appr 03d. mate Ij 5, 10, 20, 30, 50, 100, 200, 400,
and 500 ciw.
6.
Constriction pipette, wide tipped, filmed with
Parawax, approximately 100
CHKIW
7.
Small stirxlng rods*
8.
Syringe pipettes adjusted to deliver 1 ml and 3
ml quantities.
9.
10.
Transfer pipettes.
Racks suitable for holding the necessary nuiaber
of both optical tubes and 6 x BO vm tubes.
11.
Para film.
12.
Xneubators adjusted to 37oc, 99©-lQ0OC, and
158O-160OG.
13.
Refrigerated centrifuge uitti automatic timing
device.
14.
Farrand Fluor ©meter •
15.
Electric motor ©quipped ^Lth rod for mixing the
contents of the tubes (buzzer)*
17
16*
Beckman Spectrophotometer, Model DtJ, with microattachment obtained from Pyrocell Corporation.
Reagents
1*
Potassium osalate, l,-6 per cent,
Made daily from 8 per cent stock solution stored
in refrigerator*
2»
Srichloroaceti© acid solutions, 5 per cent and
13 per cent,
100 gm of redistilled trichloro-
acetic acid was dissolved and made up to 100 ml
with redistilled water*
5 per cent and 13 per
cent trichloroacetic acid solutions were prepared
from the 100 per cent solution and stored in the
refrigerator,
3,
Sodium chloride solution, 1 per cent.
4.
Dipotassium acid phosphate solutions, 4 M, 2,4 M,
and 0..16 M*.
Powdered K2HIPO4 was washed with
ethyl alcohol appros&Matelj 8 to 10 times and
then with acetone 8 to 10 times, until the
fluorescence of the acetone poured off the powder
measured no more than 10 compared to fluoreseein
standard D set at 80.
The washed K2HFO4 was al-
lowed to dry (9).
4 M K2BPO4 solution was pre-
pared as follows:
174,18 ©a washed I2HPO4 was
dissolved with redistilled water and mad© up to
S50 ml with redistilled water.
2.4 1 and 0..16 £9
18
KgHP04 solutions. veXL& prepared from the 4 M
KgHP04 solution and stored in the refrigerator.
5.
Sulfurie acid, 4.5 H and 7.0 Bk
6.
Phosphate reagent*
0.3 gm of pouder, consisting of 5 per cent
MagS0g» 94,3 pef cent IaHS0gt and 0.7 per cent
1,4-amino naphthol sulfonic acid, was dissolved
in 45 ml of redistilled water to which 5 ml of
2.5 per cent asanonitaa moigrMate in water was
added.
This phosphate reagent was freshly made
every two weeks.
7.
Perchloric acid, 70 per cent.
8.
Phosphate standard.
10 ml KHgP04 stock solution?
W&Q
680 mg of KHgP04
made up to 500 ail «& th 4.5 I HgS04.
0.5 Mi KHgP04 working standard solution:
5.0 ml
of stock solution was diluted to 100 ml with
4.5 H H2SQ4,
50 eaaa of this solution contained
0.015 01 phosphorus.
9.
Pluorescein standard solutions.
Fluorescein stock solution:
10 mg of fluorescein
was dissolved in 5 ml of 95 per cent alcohol and
about 50 ml of 0.1 1 sodiu» acetate-acetic acid
buffer solution of pH 4.5 and then made up to
volume in a one liter volumetric flask with 0.1 1
19
sodiua ae©tate-ac©tic acid buffer solution of pH
4.5..
FluorQScein working; Standard A:
10 cmm of stock
solution was made Up to 10 ml with 0*1 1 sodium
acetate-acetic acid buffer# pH 4.5.
Pluorescein \7orking Standard D«
100 cmm of solu-
tion A was made up to 1 ml with 0.1 H sodium
acetate*acetic acid buffer, pH 4.5.
All thes© working standards were placed in optical
tubes and capped with waxed stoppers.
10.
Riboflavin standard solutions.
Rib.oflavln stock standardt
20 mg of crystalline
(trSP) riboflavin was dissolved and diluted to
1000 al with 0.01 W HOI.
This stock standard has
a concentration of 0.020 mg of riboflavin per ml.
Internal & tandard of riboflavin8
1 ail of ribo-
flavin stock solution was mad© up to 100 ml with
0.01 1 hydrochloric acid solution.
5.0 caia ©f
this internal standard solution contained 0.001
meg of riboflavin.
11.
Sodium acetate-acetic acid buffer pH 4*5.
55 ml of glacial acetic acid and 66.64 ga sodium
acetate were made up to one liter with redistilled
water.
20
IS.
Hydrochloric acid, 0.01 M.
I3*
Sodium hydrosulflte solution.
0*6 gm sodium h^rdrosulfite dissolved in 5 ml of
freshly prepared 5 per cent sodium bicarbonate
in water*
This reagent was »ade up immediately
before it was to be used and kept in an ice bath
during use*
this solution is stable for only SO
minutes•
Method of Analysis of Free plus Flffl and Total Rlboflavin
lia Blood Fractions
Collection and isolation of blood fractions.
To
minimise destruction of riboflavln by light, blood eol^
lections were made in the dark with the aid of red lamps.
The subjects pr^par^d for blood collection by warming
a hand under hot tap water for several minutes.
Blood was
collected into several small tubes after a finger puncture
was made using a Ho. 11 Bard-Parkej? blade.
With the use
of a pas^affin-lined pipette © aliquots of 100 exam of fresh
blood were pipetted into separate $ x 50 mm tubes containing 500 ctm of 1.6 per cent potassium oxalate.
The mix-
ture of blood and potassium oxalate was stirred gently
with a footed stirring rod to insure an even distribution
of the red and white cells.
The measurement and mixing
were carried out as rapidly as possible.
21
USie samples t?©re then eentrifuged at 450 r.p,m. for
14 minutes at 0oG and the centrifuge t7as allor/ed to coiae
to a stop smoothly,
©le turbid supernatant which con-
tained the uhite cells was transferred i&th a fine-tipped
pipette to another tube of the same size, taking care to
draw up as few as possible of the red cells.
close to the red cell layer was avoided.
The solution
The supernatant
which contained white ceils waa usually recentrifuged at
the s&ae speed for the same period to separate any red
cells accidentally transferred.
The supernatant which
contained white cells was transferred to another tube and
centrifuged at full speed for 20 minutes at 0oC.
The
clear supernatant fluid which did not contain any white
cells was discarded by gentle suction leaving only whit©
cells in the tube.
The supernatant solution remaining in the original
tube after the white cells were drawn off,- together with
the upper portion of the red cell layer, was discarded by
gentle suction using a fine tipped pipette.
The resaaining
red cells were packed by centrifuging at full speed for an
hour In the refrigerated centrifuge.
Any further super-
natant was removed by gentle suction.
For preparing serum samples, approximately O.S to 1.0
ml of blood was alloiied to stand at room temperature for
at least 30 minutes before centrifuging the sasaple at full
22
speed at 00C for 20 minutes.
CMR
Triplicate allquota of ©0
of serum were stored at *50P» if immediate analysis
was incmavmiessit.
Method £f malysia of aerum.
friplicat© aliquots of
50 cram of serum were well mixed with 1.0 ml of 5 per cent
trlehloracetie acid in a S ml tube.
The tubes tirer©
allowed to stand for 15 minutes in an ice bath.
Then the
contents of each tube were carefully mixed using the
buzzer and the tubes were centrifuged at full speed for
10 minutes at 0oC.
Two 400 cmm aliquots of the super-
natant of each tube were transferred to separate optical
tubes, the first for th© measurement of free plus fM
ri bo flavin and the other for th© measurement of total
riboflavin.
In the first tube the 400 e» of filtrate
neutralized with 100 cmm of 2.4 M K2HPO4.
%IB.B
On© to two
hours after neutralization the fluorescence of free plus
FM rlboflavin was determined using th© Parrand Fluorometer.
M. initial reading* R^,, was mad© against
fluoresceln standard B set at 50.
A second reading, %#
was made after adding 5 cmm of the internal riboflavin
standard.
The blank reading, H3, was determined by meas-
urssient ©f fluorescence after adding 5 cam of sodium
hydrosulfite solution to th© samples.
Great care was
employed to mix all solutions thoroughly by tapping with
the finger*
Triplicate reagent blanks of 400 eiam 5 per
cent tri chloroacetlc acid were analjzed along ivith samples.
The filtrates in the tabes for measurement of total
riboflatln were allowed to hydrsljse in the dark at S7oc
for 20 hours.
All the tubes were well capped uith para*
fil® in order to avoid contamination and evaporation.
At
the end of the incubation period* the tubes were taken
from the oven and cooled at room temperature*
The con-
tents were thefe neutralized with 100 cwm. of 2*4 M K2HPO4
and the amount of total riboflavin was measured as
described for free plus MM riboflavin measurements.
Method of analysis of red cells...
Duplicate all quota
of 20 csrni of red cells were transferred into 1 ml of 1 per
cent sodium chloride solution in 8 ml pyrex tabes*
(These
suspensions were well mixed and stored frogen when
iraaaediate analysis was inconvenient.)
The red cell sus-
pension in 1 per cent laCl was well mixed with 3 ml of
IS per cent triehloroaeetic acid and allowed to stand SO
to 60 minutes in the refrigerator*
At the ©ad ©f thia
period* the samples were mixed again by using the bugger
aid centrifuged at full speed for 10 minutes.
Triplicate
400 cvm aliquots of the clear supernatant from each sample
tube were transferred to two sets of optical tubes.
The
first set contained 100 enm of 4 1 K2HFO4 for free plus
§4
HM riboflavin determinations, while th© second set was
used for total riboflavin determinations*
After a lapse
of on© to two hours* fluorescence in the first set that
had been neutralized with 4 M K2HPO4 was saeaeured for free
plus MM riboflavin as described for seruiru
feiplicate
reagent blanks wer©^ analyzed along with samples*
The allquots for total riboflavin determinations were
allowed to hydrolyee in the dark at S70C» for 20 hours*
fhe hydrolyzed samples were neutralized with 100 cmm of
4 M KgHP04 and total riboflavin was measured as described
for serum.
Method of analysis of tfliite cells.
Six allquots of
wMt© cells were analyzed for total riboflavin for each
sampling period.
The tubes containing th© luhlt© cells were
buzzed to give an even distribution of the cells.
The
cells were deproteinized by adding 110 cma of 5 per cent
trichloroacetlc acid to each tube and agitating gently,
(fhe samples could be frozen at this stag© if isaaediat©
analysis was inconvenient.)
fhe deproteinized samples
were allowed to stand at room temperature for SO minutes
to one hour.
At th© end of this period the samples were
mixed carefully again.
After centrlfu^Lng at full speed
for 15 minutes, an aliquot of 100 cam of the supernatant
was transferrred to an optical tube and allowed to •
25
h^droljg© at B^0G for 20 hours*
At the ©nd of the 20 hour
period, the tubes were taken from the oven and cooled at
rooa temperatur©*
The hydrolyeed filtrates were then
neutralized with 400 enm of 0.16 per cent K2HFO4 and rlboflavin uas measured as described for sefwm*
friplicate
blanks of 100 cum of 5 per cent trlchloroacetie acid were
analyzed along witfe the samples*
Due to the limitations of the amount of blood available* only triplicate aliquots of white cells tier©
analyzed for free plus FW riboflavin*
fhe evenly dis-
tributed whit© cells were deproteinized with 5 per cent
trichloroacetle acid and extracted as described for total
riboflavin analysis.
An 100 cam aliquot of the super*
natant of each sample was transferred into an optical tub©
and the filtrate was neutralized ismediately by mixing
with 400 em of O.IS per cent KgHPO^.,
The concentrations
of riboflavin were measured m described for serum*
The
remaining supernatant ©nd residue from both total and free
plus FMH riboflavin analyses were reserved for phosphorus
determinationsj> which were used as measures of the weights
of white cells and platelets*
Measurement of phosphorus in tiihite cells*
fh© acid-
insoluble phosphorus, together wit& the acid-soluble
phosphorus of the small ©mount of remaining supernatant.
2©
of each saaple was measured.
She app^oximatelf 10 emm of
remaining supernatant and the residue of each tube were
mixed by gentle buzzing.
added to each sample.
Tuenty twm of 7 1 H2SO4 was
After mixing, the samples were heated
in an oven for two hours at 98° to 100oG.
At th© end of
one hour the tubes were tafeen out and gently rotated to
wash down any sample adhering to the sides of tubes.
Triplicate blanks of 30 cnsa of 4.5 1 H2SO4 and standards
of 30 cam of 0»5 ng M2PO4 were treated in the same way.
After heating, the tubes -©ere cooled in a desiccator.
this point the tubes could stand over night.)
(At
Next, 10
omm of 70 per cent perchloric acid was added to each tube.
The contents in the tubes were miKed by rotating and ashed
in an oven at 158° to i60oG for two hours.
cooled in a desiccator.
The tubes were
Two hundred cwm. of phosphorus
reagent was added to each tube and the contsnts were
vigorously mixed by bugging.
After 15 minutes and after
SO minutes the tubes were buzzed again.
At the end of the
50 minutes the density of color developed was measured in
the Beekman Spectrophotometer at 690 tap.*
Triplicate
blanks and six standards were determined along with
samples..
Standards were read in triplicate before and
after th© samples.
21
The preparation of control asaaples for blood fracMiORS.
Triplicate allquota of control samples of fee respective
blood fractions were analyzed with each series of samples*
This procedure served not only to check on the reproducibility of th© analytical procedures* but also confirmed
the stability of the sample preparations which were stored
at -5°P*
For the preparation of th© white cell and red cell
control samples, approxiiaately 30 ml of vesaous blood was
used*
Two isl all quota of i?hol©, blood wer© pip©tt®d as
rapidly as possible to avoid clotting into 10 ml of 1*6
per cent potassium osalat©*
The tubes were inverted one©
©nd eentrifuged for 15 minutes at 450 r«p«m.
The super-
natant which Contained whit© cells was pooled in a beaker.
Aliquots of 400 cam of supernatant were transferred into
6 x 50 cma tubes.
Constant stirring was maintained during
sampling to giv© as even a distribution as.possible of
white cells in the supernatant*
She white cells were
packed down by centrifuging for 20 minutes at full speed
at 0oCc
The clear supernatant was removed by gentle sue*
tion and 110 cms. ©f 5 per cent irlehloroacetic acid wasadded to each tube*
The contents were nixed by bussing
and capped with parafilm and stored at -§0F*
For the red cell control sample, th© remaining red
cells> after the supernatant viaioh contained whit© cells
28
was dram off, u©r© packed by centrifuglng for an hour at
full speed at 00C,
triplicate aliquot© of 300 csam of
packed red cells *7©re pipetted into 10 ^1 of 1 por cent
laCl and laiseed well.
TMrty ml of IS per cent trichloro^
acetic acid was added to the misctur© of laCl and red
cells*
The mixture was placed in the refrigerator for SO
to 60 minutes*
At the end of this period, the mixture
was stirred again by using the buszer and centrifuged at
full speed for 10 minutes*
fh© filtrates from t&te three
preparations were pooled and stored in a glass-stoppered
brovm bottle in the refrigerator*
For the preparation of control serum samples» approM*
mately 10 ml of whole blood was allowed to stand for 30
minutes at room temperature and then eentrifuged for 20
minutes at full speed at 0oC.
in a beaker.
The clear serim was pooled
Aliquots of SO cram of serum were pipetted
into tubes which were capped with parafilm and stored at
Calculation of Results
The calculation of free plus Fffl and total riboflavia
concentrations required corrections for dilutims due to
the addition of the internal standard and reducing solu*
tion during fluorescence measurements as well as for dilutions in preparation of filtrates of the samples.
The Bg»
20
HvLQiPQSGmeQ ^©^lag ©f .sompX© ptmi iiat©Bial sfeaaiaapd^ smel
%, i?©clme©$ flmo^oeie*^©© roadisg, o©r0 dd?r©etei to ig*
©mi Bg1» faep^eti^rely*
Sa© lg« tia© eaieiiltafe©*! a@ mo p&
&<mt ®i Sg pitas fig (5*0 csm ©f SBtoifial e^aadard ai<t©t to
i00»O twm c& eolufelfia) ©ai %' ma os&euSL&tod as 3 pos*
cent «ri? lg ^lus % (S#© ens of ^Mmelag ag<»t ©&#©$ to &©
soltttlon affes5 the fssilsg ■%)»
£3eg B£bon«^
Si -Ha*
tip"-
Sg*
%^
- -r—B-
--,-,—.- - -^--^j**^.^^
9 Goareoetod ^luopoaoeneo of &Q%le ♦
Ctg * ©«01 Eg)
C% ♦ o.oe %}
CsAottla^Lcn. fo^ r©€ ©©IS,©*
to
©s- total)
♦ 0*01 Bs)
43
C% * 0.@g Rg)
alitm©^
30
Calculation for ■giiite cells*
fhe samples analyzed
for phosphorus contained all of the acid insoluble phosphorus and, with the specific calibrated volumes of
pipettes used in these analysess 8 per cent of the acidsoluble phosphorus (8*4 cnp remained of the original 108*6
cam of filtrate after removal of 100*2 cm& aliquot).
Since white cells contain an average of 33 aslcromoles of
acid-insoluble phosphorus and 28 microinoles of acidsoluble phosphorus per gja of white cells (7, ,p# 457-470),
the samples contained 33 plus 0.08 x 28 which equals 35*2
aieromoles of phosphorus per ©a of white cells*
There-
fore, the meg of riboflavin per 35.2 micromoles of phosphorus found were numerically equal to the meg of riboflavin per ©a of white cells.
Meg Riboflavin
per 100 gm
£
meg riboflavin in entire &
S-QQ
x 35.2
sample (free * FM or
yUM Of P
total)
leg Riboflavin
in entire.
meg riboflavin
in aliquot
Meg Riboflavin 6
in aliquot
"
B^ - Bg'
Rgi-'H,
whares; R^
*
Rg' s
R3' 2
x
r©l. used of TCA
filtrate of TOG
meg of riboflavin
(internal standard)
Pluoreseence reading of sample
Corrected fluorescence of sample plus
internal standard (Rg * 0.01 Rg)
Corrected reduced fluorescence
(Rg t 0.02 Rg)
jm of P a m V in standard x Density of sample
/.™_
A—
Density of standar<
31
Calculations for actual free plus FM and FAD
riboflavin jn bipod fractions*.
Since in this method FAD
riboflavin before hydro lysis is about 14 per cent as
fluorescent as riboflavin, free plus WM and PAD riboflavin coneentrations in blood fraction© were calculated
as follows?
f
„
Total
Apparent
Free
»
FM
riboflavin
PAD riboflavin * ^riboflavin,
0,86
1"
Actual free
plus FM
riboflavin
"-■
•
'■
■'■"»»..»,HI .l.i. i.iir
i
.'■
Apparent Free * FM riboflavin
0*14
FAD
For some samples the concentrations of Free plus
riboflavin were zero-.
Small smoUnts of free plus H®
riboflavin were measured* but feey were calculated as
being due to FAD riboflavin*
Heproducibllity of Method in This Investigation
The mean concentrations.of total riboflavin determined from triplicate aliquots of control samples measured
with each series of blood fraction analyses are summarised
in Table 4.
Fourteen series of samples were determined
because two test days were involved for subjects BEH and
SJM*
32
fable 4.
Concentrations of Total Riboflavin Detenalned
in Control Samples of Tlbapee Blood Fractions
Sample Series
Serum
led Cells
I'Jhite
mcg/100 ml
meg/100 ml
mcg/100 gp
BB1 1'1-29*69)
3.7
14.4
190
JOF 1[2- 3-59)
3,8
16.0
190
mm {:2*10«59)
3.9
15,9
165
MLP 1[2-17-59)
3.9
14.8
181
sai i'2-21*29)
3.8
20.1
168
1113 1[2-26*59)
4.2
15.5
157
EB¥ j S- 5-59)
3.7
13.5
163
am i'5-
8-59)
3.7
15.3
180
ESSB 1(3-12-59)
3.6
16.1
192
EA@ j[3-17-59)
3.1
15.3
162
QT 1 3-19-59)
2.9
15.8
197
9*59)
3.3
14.3
199
sw «'4-14-59)
3.5
14.1
198
BEB 1[4*21-59)
3.2
15.3
176
sm <[4-
Q^LIB
S3
BSSUm MB DISCffSSOT
Th© concentrations of fr©© plus flavin mononucleotid©
)$ Tlavin adenine dlnucleotM© (FAD) and total rlboflavin in blood fractions following a 2 mg test dos© of
riboflavin ar© susMariged for tuelv© subjects in fable S.
All concentrations reported for serum represent the m©an
of ttxr©e samples*
Values determined for th© samples in
general agreed efttMn 0 p©r cent? th© masiauffi variation
tj&s 10 per cent.
All concentrations reported for r©d cells
ar® th© mean of values for six. aliquots* triplieat© allquots of ©aoh of ti7o red cell samples.*
In gmeral the
values determined agreed within 10 per cent? th© aasiausi
variation was 18 per cent.
For dait© cells free plus fW
as well as total riboflavin concentrations* allowing cal*-.
culated FAD ribofl&vin eaacentrations* were determined for
only five of the twelve subjects.
¥alues reported for
whit© cells represent th© mean of four to sise samples for
total and two to three samples for free plus WM riboflavin.
Because of the larger variation in concentra-
tions among aliquots* the standard errors of the means
were calculated (Si* p* 36) and are included in fable S.
Greater variations among values determined in whit© cell
analyses are due to the small sis© of samples m.&t as
described in th© method,' th© determination of th© concen*
trations of riboflavin in whit© cells involves two
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34
35
proceduress
deterMning the concentrations of the
fluorescent substance and measuring the amount of the
phosphorus in order to estimate the weight of whit© cells*
Changes in Concentrations of.Riboflavin in Blood
Fractions After the Test Dose of 2 mg of IJiboflavin
The Concentrations of Riboflairin jia Serum
A similar general pattern of changes in concentra*
tiong of total and free plus BW riboilavln ih serum for
all subjects was found, Figures 1 and 2,
The hi^est con*
eentrations for free plus Hffi and total riboflavin xiere
measured for all twelve subjects at one-half hour after
th© test dose was given*
Th© de^ee of .rise, however,
from fasting value to the value at one~iaalf hour varied,
with individuals.
Acs evident frora Table 5, increases ©f
approximately 200 to 1400 per cent were observed for free
plus Mi riboflavin#
Increases in total riboflavin were
approximately 60 to 180 per cent of the fasting values.
At th© end of three hours concentrations of total riboflavin had returned to fasting levels for four subjects,
but for two of them concentrations again were elevated at
the third hour. .For the eight other subjects total riboflavin concentrations were decreasing toi?©rd fasting
levels at the third hour.
Concentrations of free plus
FES® riboflavin had returned to fasting concentrations for
36
FIGURE!. TOTAL RIBOFLAVIN CONCENTRATIONS
IN BLOOD FRACTIONS FOLLOWING A
2MG TEST DOSE OF RIBOFLAVIN
YOUNGER WOMEN
OLDER WOMEN
WHITE CELLS
240
5
220 .
o
o
-
200
tt
o
5
o
o
hi
O
2
I
2
HOURS AFTER TEST DOSE
MEN
37
FIGURE 2.
FREE PLUS FMN RIBOFLAVIN CONCENTRATIONS
IN BLOOD FRACTIONS FOLLOWING A 2M6 TEST
DOSE OF RIBOFLAVIN
YOUNGER WOMENOLDER WOMEN-
80 .
WHITE CELLS
MEN
3
O
O
I
CO
RED CELLS
©
o
SERUM
I
2
HOURS AFTER TEST DOSE
38
FIGURE 3.
FAD RIBOFLAVIN CONCENTRATIONS
IN BLOOD FRACTIONS FOLLOWING A
2MG TEST DOSE OF RIBOFLAVIN
YOUNGER WOMEN
OLDER WOMEN-
210
WHITE CELLS
r
o
o
S
CO
5
no
RED CELLS
o
o
IB
to
£
I
2
HOURS AFTER TEST DOSE
MEN
S9
flv© subjects duatdng the thre© hours; concentrations were
decreasing toward fasting concentrations for the others*
Changes in F.feB riboflavin concentrations in semrn
were smaller and raor® irregular thai changes in the free
plus MM riboflavin. Figure S*
Increases of 20 per cent
or more of fasting values occurred in seven subjeeta,
how&v®?,
fhe most marked increase-, approximately 100 per
cent, in;as cbserved for one man, MM, who reached maximum
concentration at the second hour*
lo consistent differ-
ence in patterns of change was apparent between the group
of three older women ©nd the group of sis younger wcsnen or
between men and uomen.
The Concentrations of Hiboflavin in led Cells
The changes in concentrations of riboflavin in red
cells showed greater indivMual variation than the changes
in serum. Figures It 2» and 3.
All but on© subject
responded to the test dose nith an increased total rib©*
flavin concentration at one-TisOLf hour, although soiae in*
creases itrere very small, fable 5*
Ba© highest concentra*
tions of total riboflavin in red cells were measured at
fasting for one subject, at one-half hour for four subjects 5 at one hour for four subjects, and at two or three
hours for two subjects*
For one subject, SB?, concentra-
tions were only slightly but equally elevated from fasting
40
at one-half through the, second hour.
Prom a slight de*
crease in en© subject saasdarum increases up to ©pproxiraately 50 per cent were observed*
In six subjects* i&m.
values at the end of three hours were considerably highor
than the values at fasting..
Changes in concentrations of free plus FBM ribofl&vln
in red cells were smaller than eh^iges in cone©ntratiana
of FAD riboflavin, .Figures 2 and 3.
From a slight decrease
in one subject maximum increases up to SO per cent for
free plus FM riboflavin in serum were observed..
From
decreases in FAB concentrations for two subjects maximum
increases up to 45 per cent were observed.
Although, in
general, total riboflavin eone©ntrations in red cells were
somewhat lower for t&e older women and the men tfoan those
of the younger women,, the older women and the w&n generally
maintained higher free plus FM riboflavin eoneeatratlona
than the younger women*
In ecsitrast* therefor©, th© group
of six younger women generally maintained higher CGncen*
tratiCEfis of FAB riboflavin in red cells than the other
two groups.
The Concentrations of Hiboflavin in tfhite Cellsi
In spite ©f th© wider ranges of values for aliquota
of whit© cell ssmples, increases from fasting eoncentra*
tions of total riboflavia were significant for all but
41
three, JOP, SJE9» snd. EA©#
Statisticelly aigalfleant
differences between concentrations at ©ay one hour and
the ccaacentratians at the preceding hour, measured by
Fisher's "t" test (21, p. 65), are indicated at the 5 per
cent .(*) and 1 per cent (**) levels of probability in
Table 5.
After the 2 mg test dose* the highest concentrations
of total riboflavin in white cells were observed at onehalf hour for 8 subjects, at one hour for 2 subjects, and
at two hours for one subject*
One subject, BEH, had an
equal elevation from fasting concentration from one-half
through two hours.
Increases of 4 to 25 per cent in the
ooncentratLons of total riboflavin in white cells were
observed.
In general, the concentrations of total ribo-
flavin in white cells returned toward or were lower than
fasting levels at the end of three hours*
As shown in
Figure 1* the group of three older women and the group of
three men Bjaintained more consistent patterns in changes
of total riboflavin in white cells than the group of six
younger women.
In the five subjects for which data are available,
the concentrations of free plus MM and PAD -riboflavin in
white cells followed irregular individual changes in concentrations.
However, both free plus Pffli end PAD
riboflavin in whit© cells increased in most subjects after
42
th© test dose but to a much greater degree in the free
plus FM fraction.
Changes from fasting mlues of free
plus MM rihoflavin varied from slight decreases in two
subjects to a maximum of over 400 per cent and of FAB
varied from a slight decrease in one subject to a maximum
of 25 per cent.
Discussion
fhe concentrations of free plus FEIft* FAD, and total
riboflavin in each blood fraction increased in most subjects after a test dose of 2 mg of riboflavin, althou^ii
there was considerable individual variation.
Changes in total riboflavin concentrations in serum
were similar to those reported by Burch et aU (7, p. 45,7*
470) and Wu, Warren, and Storvlck (27, p. 231-240).
Changes in total riboflavin concentrations in red and
white cells were generally greater than those determined
in a preliminary study in this laboratory. Table 2.
In general, total riboflavin concentrations in blood
fractions were returning toward fasting values within the
three hours after the test dose.
This indicates that
riboflavin is not readily stored in blood serum or cells.
Klein and Kobn also reported that tshen the administration
of riboflavin was discontinued, the increased riboflavin
level returned to its original value (16, p. 177-189).
43
It has been reported that there are no special organs
where riboflavin is stored (11, p. 1157; 15, p. 514-516).
Bro-Rasmussen (6, p. 1-23), in his recent review, reported
that liberation of free riboflavin fro® the tissues
shortly after the intake of the vitamin was caused by a
low renal threshold for riboflavin*
fhe changes in PAD riboflavin concentrations which
occurred in the various blood fractions were of particular
interest*
Changes in the proportion of FAD perhaps
reflect chemical reactions involving riboflavin.
For
their five subjects, Wu et al. (27, p* 231-240) reported
that after reaching the highest serum concentrations following a 2 mg riboflavin test dose free plus POT riboflavin returned to fasting levels more rapidly than total
riboflavin concentrations,
were increasing.
PAD concentrations apparently
In 1940, Klein and Kohn (16, p. 177-189)
had reported that the ihgestion of 200 mg of riboflavin
by mouth caused approximately a SO per cent increase in
the FAD content of blood cells*
In this experiment 2 mg
of riboflavin caused increases of FAD concentrations in all
blood fractions in some subjects, but most consistently in
the red cell fraction*
Maximum increases of concentrations
of FAD were approximately 100 per cent in serum, 45 per
cent in red cells, and 25 per cent in white cells*
It is
possible, however, that maximum changes in FAD riboflavin
44
may not have occiirred within the three hour period of
these experiments,
Ccacentrations of FAD were increasing
from the second to third hours in serum for 6 subjects,
in red cells for 3 subjects* and in white cells for 1
subject*
Comparison of T0o Levels of Test Dose
One subject, BEH, was given two different levels of
rlboflavin as test doses* 2 mg and 5 mg on two different
days*
Concentrations of rlboflavin in all blood fractions
were determined at fasting and one»half, one* two* and
three hours after each test dose*
Total rlboflavin in all
fractions increased from fasting concentrations after both
test doses. Table 6 and Figure 4*
The highest concentra-
tions of total rlboflavin were measured at one*half hour
after both test doses in serum and in whit© cells*
In the
red cells the highest concentrations of total rlboflavin
were measured at one hour and at three hours* respectively* after the 2 mg and 5 mg test doses*
Changes in
total rlboflavin in serum and tSiite cells were much
greater following the S mg than the 2 mg test dose*
Klaxl-
mum increases were approximately 250 and 50 per cent In
the serum and white cells* respectively* after the 5 mg
test dose of rlboflavin, whereas only 100 and 10 per cent,
respectively, after th@ 2 mg test dose.
In the red cells.
fab;L© 6... Riboflavlzi Conceatra fcims i n Blood Fraeticsns
Follooing Two Levels of Tea t^Dose for BH
ged. eelIs
Serum
Lfce cells
Hour
PAD
"fotai1 Kee*FES
Fm
Fm
Free*Fffl
Tetal
meg/
meg/
meg/
meg/
meg/
meg/
meg/
meg/
meg/
100 ml 100 mH 100 al
100 ail
100 ml 100 ml 100 gm
100 gm
100 gm
2 mg Test Dose
F
A
3
1
2
3
2.6
7.3
5.2
4*7
4.0
1.9
1.5
1.4
1.S
2.2
4.5
8.8
6.6
6.0
6.2
5.3
5.7
5.9
5.4
4*6
15.7
17.3
15.3
16.3
17.0
21.0
23. 0
21.2
21.7
21.6
2.0
0*5
1.7
1.7
1.9
4.4
14.4
12.1
10.2
7.4
3.5
3.7
2.9
• . 4.2
5.1
16.0
17.9
19.4
16*5
16.2
19.5
21.6
22.3
20.7
21.3
20427 X
227X7 *2
227«5
22642
20843 **
5 mg T©st Dose
P
1
2
S
2.4
14.1
10*4
8.5
5.. 5
1
Standard error of ttoe mean.
2
* a P^fS-OR*
'
23
82
19
61
10
164
190
194
171
241
187X5
272*17**
21342 **
232*6 **
251*15
46
FIGURE 4 TOTAL RIBOFLAVIN CONCENTRATIONS IN
BLOOD FRACTIONS OF BEH FOLLOWING
TEST DOSES OF RIBOFLAVIN
WHITE CELLS
280,.
- 2 MG TEST DOSE
- 5MG TEST DOSE
RED CELLS
24
20i
SERUM
o
£/«
o a
3
£
/
2
HOURS AFTER TEST DOSE
■i
47
however, no significant difference in the degrees of increase in total riboflavin was found following the two
test doses*
An increase of approximately 10 per cent from
fasting level to the highest concentrations of riboflavin
in red cells was observed in both tests*
Comparison of Duplicate Tests
A test ms made to determine whether the changes in
concentrati ons of riboflavin in blood fractions in a
single Individual tended to follow a constant pattern.
Subject
SJ1
was selected because of the marked differ*
ence in the response to the test dose of this subject compared to the others*
The second experiment was made two
months later than the first*
The results of these two
esperimaits are presented in Table 7.
The changes in con-
centrations of riboflavin in serum ivere similar*
The de-
gree of increase from fasting levels to the highest concentrations differed, however*
The percentage increases
were approximately 3S0 and 280 per ceat for fre© plus WM
riboflavin and 80 and 110 per cent for total riboflavin*
The changes in concentrations of riboflavin in red and
Aite cells differed greatly on the two days.
Changes in
concentrations of riboflavin following a test dose do not
appear to be constant* particularly in the cell fractions*
03
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64
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43
Factors vshich may be responsible for these day to daydifferences are unknown.
50
SmMAM
The concentrations of free plus MB ©nd total ribo*
flavin in blood fractions at fasting and one-half> one,
two, and three hoars after a 2 mg test dose of riboflavin
were determined for twelve subjects.
The calculated
values of free plus FW and FAD riboflavin together with
total riboflavin concentrations in blood fractions are
presented.
The concentrations of riboflavin in all blood frac*
tions increased in most subjects after the test dose*
It
was of interest that the eoncentr&tions of riboflavin
changed not coaly in the forsa of free plus MM but also in
the PAD riboflavin fraction*
Maximum increases of free
plus MM, FAD and total riboflavin were as follows:
1400,
100, and 180 per cent, respectively, in serum; SO, 45, and
30 per cent, respectively, in red cellsj and 400> 25, and
25 per cent, respectively, in white cells*
At the end of
three hours the cmeentrations of free plus FMH and total
riboflavin in serum and white cells generally had decreased
totmrd or were lower than fasting levels.
Xn red cells
the concentrations of total riboflavin were considerably
higher than the values at fasting for several subjects*
The FAD riboflavin concentrations at the end of three
hours ivere higher than fasting levels in serum and white
51
cells for a few subjects but were elevated in red cells
for seven subjects.
In this small group of subjects age or sex seemed to
have little influence on the cone ©it rations of riboflavin
in blood fractions.
However* th© group of siz younger
women in general maintained higher concentrations of F&D
riboflavin in red cells than the older women or men*
In one subject tested, concentrations of riboflavin
in serum and in white cells were higher following the 5 tag
than the 2 rag test dose,
fhe size of the dose made no
difference in the concentrations of riboflavin in red
cells.
In a comparison of duplicate tests in one subject
using 2 mg doses of riboflavin, concentrations of riboflavin in serum were similar but the times at which
changes occurred in concentrations in red sad white cells
differed greatly on the two days.
52
BIBLXOOBAm
1.
Axelrod, A* D*, T. D» Spies and C. A, Elvohjemu
Riboflavin content of blood and BJUSCI© In normal
and In malnourished humans. Proceedings of the
Society for Experimental Biology and Medicine
462146-149. 1941*
2.
Bartlett, Mary M. Red blood cell niacln and plasma
riboflavin levels in a group of normal children.
Journal of Nutrition 57j 157-168* 1955.
5.
Bessey, Otto A»# Oliver H. Lowry and Kuth H. Love.
The fluorometrle measurement of the nucleotides
of riboflavin and their concentration in tissue.
Journal of Biological Gheadstry 180:755-769.
1949.
4.
Bessey, Otto A** 1* K. Horwitt and Ruth H. Love.
Dietary deprivation of riboflavin and blood
riboflavin levels in man. Journal of lutrition
58:367-383. 1956.
5.
Bradford, Slid A. 1.. md KU Coke* Observations on
the microbiological determination of riboflavin
in blood. Biochemical Jouraal 39:379-385. 1945.
6.
Bro-Rasmussen, Finn. The riboflavin requlremits of
animals and man and associated metabolic relations. I. Technique of estimating require*
ment, and modifying circumstances, nutrition
Abstracts and Eevlews 28*1-23. 1958.
7*
Burch, Helen B., Otto A*. Bessey and Oliver B* Lovjry.
Fluorometrlc measurements of riboflavin and its
natural derivatives in small quantities of blood
serum ©nd cells. Journal of Biological Ghemlstry 175:457-470. 1948.
8.
Burch, Helen B., et al* lutrition resurvey in
Bataan, Philippines, 1950. Journal of nutrition 46:239-254* 1952.
9.
Edwards, Margaret• Unpublished research on total
riboflavin in Tshole blood snd serum. Corvallis,
Oregon, Agricultural Experiment Station,
lutrition Laboratory. 1956.
5S
10.
Frutcaa, Joseph S. and Sofia SiHraonds. General
Mochemistry. 2d ed. Mew York, Wiley, 1958.
1077 p.
11.
Hawk, Philip B., Bernard X;* Oser and William H*
Summersem. Practical physiological chemistry*
ISth ed. Philadelphia, Blaklston, 1954. 1325 p.
12.
Horwitt, M. K* Rihoflavln. I?. Biochcdaleal systems.
Ins W. H. Sebrell and R. S. Harris' ^Ehe vita*
rains. Vol. S. Meu York, Academic Press, 1954.
p. 334-357.
13.
Eegsted, D. M. Hiboflavin. VIX* Estimation, tnz
W. H. Sebrell and R. S. Harris* The vitsmins.
Vol. 3. leu York, Academic Press, 1954.
p. 361-373.
14.
Irgens-Miller, Ida. Unpublished research on total
riboflavin in red and uhtte blood cells. 0or«
vallis, Oregon, Agricultural Bsperiment Station,
nutrition Laboratory. 1956.
15.
Jolliffe, lorman, P. F. fisdall and Paul R. Cannon.
Clinical nutrition* lew York, Paul B, Hoefoer,
1950. 925 p.
16.
Klein, Raymond J* md Henry 1. Kohn. The synthesis
of flavin adenin© dinucleotid© from riboflavin
by humsffli blood cells in vitro and in vivo.
Journal of BiologieallJhemia try 1317177-189,
1940.
17*
Morley, lina H. Unpublished research on blood fraction and urine riboflavin. Corvallis, Oregon,
Agricultural Experiment Station, lutritlon
Laboratory. 1958.
18.
lational Research Council* Recommended dietary
allowances. Rev. 1958. Washington, lational
Academy of Sciences, lational Research Council,
1958. 36 p. (Publication Ho. 589)
19.
Prager, lortcsa P., et al. Ihole blood riboflavin
levels in healtliy Individuals and in patients
manifesting various blood dyscrasias. Journal
of Laboratory and Clinical Medicine 52:206-211.
1958*
54
20*
Samvik* Ulla* Unpublished research oa riboflavin
concentrations in serum* Corvallis, Oregon,
Agricultural Experimait Station, Nutrition
Laboratory* 1954.
21*
Snedecor, George W* Statistical methods* 4th ed*
AmeSj loua* Iowa State College Press * 1950*
485 p*
22*
Strong, F* M., et al.
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