AN ANSTBACT 0' TBB TELESIS OP
Elizabeth Willard. Cox for the Ph. D. in Pood.e and Nutrition
te Thesis Presented:
August 12, 1949
The Determination of Thiamine in the Blood of HUmP't Subjects
Redacted for privacy
Abstract Approved:
or Professor
Title:
The blood thiamine concentration of 11 subjects was determined
by means of the iedemann and Kmieciak (1943) method. every 5 days
during a 30-day experimental period. Study I was conducted on 5
students in 1947 and Study II was conducted on 6 students in 1948.
The subjects in both studies were on diets in which their intake of
ascorbic acid only was controlled. A record was kept of each subjectts
food intake. The daily values for total Calories, non-fat Calories
and thiamine in the diet were obtained from food. tables. Non-fat
Calories, the ratio of non-tat Calories to total Calories, thiamine
to 1000 Calories and thiamine to non-fat Calories were calculated..
The blood, thiamine values for the subjects in Study I (all girls)
ranged from 4.91 to 10.85 meg per cent. There was a general decrease
throughout the study in. the mean intake of thiamine expressed in terms
The greater
of meg per 1000 Calories and meg per 1000 non-fat Calories.
the decrease in thiamine intake in terms of meg per 1000 non-fat Calories
the greater was the loss of blood thiamine. The values for thiamine in
the blood of the boys in Study II ranged from 8.00 to 15.32 meg per cent
The
and for the one girl in. Study II from 8.35 to 13.93 meg per cent.
blood thiamine values for the subjects in Study II did not indicate the
same relationship to thiamine intake as did. those in Study I. There was
an increase in the concentration of the thiamine in the blood even though
there was a decrease in the mean thiamine intake from the beginning to the
end. of the experiment. It would appear, therefore, that the boys obtained. sufficient thiamine throughout the 30-day period.
No data have been obtained which indicate whether or not there are
variations from day to day in. the concentration of thiamine in the blood
when subjects are maintained on a. constant intake of thiamine. A
metabolism study using 5 adult women as subjects was planned in order to
determine the daily values for thiamine in the blood when subjects were
maintained on a controlled diet for a period of 52 days. An unpublished
micro-method for the determination of thiamine in the blood developed by
Dr. H. Burob (1948) was to be used.
The experimental diet consisted. of a basal diet providing approximately 1000 Calories and 300 meg o thiamine with additions to the basal
diet planned in units providing approximately 500 Calories and 150 meg of
thiamine. The values for protein, fat, carbohydrate and total Calories
gi
-.
were obtained, from food tables.
lTon-.fat Calories were calculated. and
the thiamine coutent of the foode was determined by analysis. LU 5
subjects ate the same food. each day.
In spite of the fact that considerable preliminary work was performed. before the study started. the blood. thiamine values obtained in
the nutrition laboratory were always significantly lower than results
It was decided. to freeze the blood. samples
obtained. by other workers.
after the protein had been removed and work on certain aspects of the
method before any analyses of the blood thiamine were made.
the Burch method. included. variation in the amounts
of potassium acetate used, use of different trichioroacetic acid. reagents,
PU.rther
study of
tests for enzyme activity, variations in the incubation procedure,
variations in the procedure for the oxidation to tbioobrome and. in the
extraction of thiochrome, reading samples sooner after transfer to
optically matched tubes, variations in the irradiation procedure, use of
all new reagents, use of another miero-pbotofluorometer, development of
standard. curves and the determination of the response of two subjects to
an oral test dose of thiamine hydrocloride. 1one of these variations
resulted. in a solution of the problem,
Suggestions for future work with the Buroh method are discussed,
TEE iTFRMX}I&TIO! OF THIJMIEE IN TEE BLOOD
OF HUMA1I SUMECTS
ELIZA.BETK WILLLRD OOX
A TEESIS
submitted. to
OREGON STATE OOLIGE
in partial fulfillment of
the requirements for the
degree of
DOCTOR OF PHILOSOPHY
Augast l9i.9
APPROVD:
Redacted for privacy
Professor of Foods and nutrition
In Cbarge of Major
Redacted for privacy
Head of
partment of Foods and. Nu.trition
Redacted for privacy
hairun of School Gradugte Committee
Redacted for privacy
Dean of Graduate School
Shirley Kosko:
Typist
AC1NOWLEDGME1TS
Sincere gratitude is expressed. to Dr. Clara A. Storvick for her
constant assistance and. encouragement thronbout the thiamine studios.
The author has learned mach from her valuable criticisms and.
suggestions.
Appreciation is also expressed to Dr. Margaret L. Fincke
for her interest in the problem, careful reading of the thesis and
the helpful suggestions she has given.
Grateful acknowledgment is made to Dr. Helen B. Burch for
allowing us to work with. her npublished micromethod for the
determination of thiamine and, for her patience in answering in detail
our many questions.
The cooperation, interest and enthusiasm of the subjects and
the other workers in the laboratory lightened considerably the long
hours of research involved.
To each of them appreciation is expressed.
To Mr. C. H. Wang for checking the calibration of the micro
pipettes, to Dr. 3. 3. Christensen for help
in the redistillation
of trichloroacetic acid and to Dr. J. R. Haag for the use of some
of his preparation of redistilled trichioroacotic acid the author
expresses her appreciation.
Last of all, my thanks to Shirley Kosko who used such painstaking
care not only as a laboratory assistant bat in the typing o± this
thesis.
TABLE OF OONTENTS
Page
CHAPTER
I
. ..
REVIEW OF LITERATURE
1
METHODS FOR ASSESSING THE STATE OF NUTRITION WITH
RESPECT TO THIAMINE...
Clinical
...
examination......
Demonstration of Biochemical
.
1
or Physio1gica1
1
.
1
Physiologicaltests ..................... ....
4
Past Dietary Intake ............ ..
4
Studies on urinary excretion
Studies on blood or blood fractions ........ .
Estimation of
3
METHODS FOR TEE flETERMINT ION OF INTERMEDIARY
METABOLITES, THIAMINE AND COCARXYLASE IN BLOOD
ORBLOODACTIONS
...........
5
.
1yruvieLacticAcid.s .......
.
5
Thiamine in Plasma and Sorwn Compared with that
Q,
Whole Blood..
Thiamine in. the
. . .
6
...... . .........
Formed Elements of the Blood
7
Cocarboxylase in Whole Blood ..................
8
Thiamine inWhole Blood ........................
9
Phycomyces blakesleearnis ....................
Fermentation ................... , ............
Thiochrome ..........
9
9
10
THE QUESTION OF THE SIGNIFICANCE OF BlOOD
THIAMINE VALUES IN ASSESSING THE STATE OF
NUTRITION WITH RESPECT TO THAT VITAMIN ....... ....
CEAPTER
II
PROCEDURE
THE EXPERIMENTAL STUDY
14
WHICH
IEDEM1UThT AND KMIECIAK METHOD WAS USED FOR TUE
DETERMINAT ION OF BlOOD THIAMINE ............... .....
18
PLAN OF EXPERIMENTS ............................. .
18
TABLE OF CONTENTS (continued.)
Page
CHAPTER
II
(continued)
........
19
ONEBALDAILYPROOEDURE..............
20
DESCRIPTION OP SUBJECTS
CHAPTER III ANAMTICAL PROCEDURE FOR TEE ThIEDEMANN
ICMIECIAK METHOD ...............................
EQJUIPI4ENT USED. .............. . . ..................
21
REAGENTSUSED .................................. ..
22
DETERMINATION OF THIAMINE IN TILE BLOOD ......... ..
25
Collection of Blood Sample .................... .
25
Digestion with Phosphatase and Precipitation
theProtein ....................................
26
Adsorption
Elution ........................
28
Oxidation to Thioclirome ........................
29
DETEBMINAT ION OF STANDARD CURVE ................
CHAPTER
30
WH
IV RESULTS AND DISCUSSION OF THE STUDIES
USED
IN
KMIEOIAX METHOD
FRIEBEMANN
DETEBMINATIONOFBLOODTHIAMINE
CHAPTER
2].
............ ..
V PROCEDURE FOR THE EXPERIMENTAL STUDY IN WHICH IT
WAS PLA11iED TO USE THE BUBCH METHOD FOR THE
DETERMINA.TION OP BLOOD THIAMINE .....................
PLAN OF THE STUDY ............................... .
45
EXPERIMENTAL DIET ............................ ..
CHAPTER
VI
DESCRIPTION OF THE SUBJECTS ..................... .
5].
ICAL PROCEDURE FOR THE BURCH METHOD ....... ...
55
EQ,UIPMENT USED ................................ 0ø
55
TABLE OF CONTENTS (continued.)
Page
CHAPTER
VI
CHAPTER VII
(continued)
REAGENTS USED
57
DETERMINATION OF THIAMINE IN THE BLOOD
59
Co1leotionofBloodSaxp1e .....................
59
Precipitation of the Protein ................
59
Digest ion with Phosphatase ....................
60
Oxidation to Tbiochrome .......................
61
Calculations
62
.......................
DETERMINATION OF EEMATOCRIT .........................
6k
NEED FOR DETERMINATION OF REMATOCRIT ............
6k
EQ,UIPMENT USEI) ...................................
6k
PROCEDURE FOR DETERMINATION OF H.RMATOCBIT .......
65
SelectionofTubes .............................
6
Collectionof Blood ............................
65
Oentrifu.ation .................................
65
Caleu.lation ..................................
66
RESULTS OF THE DETERMINATION OF HEMATOCRIT
CHAPTER VIII FURTHER STUDY OF TEE BURGH METHOD ..............
NEED FOR FURTHER STUDY OF THE BURGH METHOD
66
67
67
METHODS USED FOR FURTEER STUDY OF THE BURGH
METHOD ............................................
67
Varying the Amounts of Potassium Acetate Used..
67
TABLE OF CONTENTS (continued)
Page
OKAPTER VIII (continued)
The determination of the pH of the blood
supernatant using varying amounts of
potassiumacotate ........................ ....
67
The determination of the pH of the blood
supernatant plus varying amounts of
potassium acetate and acid. phosphataso.......
68
Determination of thiamine in blood when
varying amounts of potassium acetate
were used. ............................... . . *
68
The Use of Different Trichloroacetic
AcidReagents ..................................
72
The use of red.istilled. trichloroacetio acid4.
72
Use of different brands of trichloroacetic
acid ........................................ .
73
Enzyme Aetivi..... ............... ..
73
Tests
Determination of blood thiamine with and.
without the use of clarase ...............
73
Varying the amount of olarase used ...........
74
Use of acid phosphatase for enzyme hydrolysis
74
Testing the activity of enzymes using
cocarboxylase ................................
75
Variations in Incubation Procedure ............
.
76
Using longer time for incubation ............
.
76
Using higher incubation temperature ........
.
77
Variations in the Procedure for Oxidation
Thiochrome and in the Extraction of Thiochrome.
79
Use of larger amounts of potassium ferncyanide......................................
79
TABLE OP CO1TENTS (continued.)
Page
CEAPTER VIII (continued.)
Omission of the sodium dihydrogen phosphateperoxide mixture ............................
79
Variation in the amount of sodium dihydrogen
phosphate-peroxide mixture used. ..........
...
80
Use of iso-butyl alcohol instead. of
n-butyl alcohol ..............................
82
Readin, Samples Sooner after Transfer
Optically Matched Pubes ....................... .
83
Variation in the Irradiation Procedure ........ .
83
Use of a new irradiation lamp ............... .
83
Variation in the time of irradiation .........
jReagents ............................
85
Use of Another Parrand. Micro-Photofluorometer..
85
The Determination
86
Stand
Curves. ......... .
Addition of thiamine hydrochloride to blood
CHAPTER
IX
supernatants ................................ .
86
Addition of thiamine
hydrochloride to blood..
90
Determination of a curve using thiamine
.................... .
hydrochloride alone
90
Determination of a curve using cocarboxylase
a lone ........................................
91
Addition of cocarboxylase to blood ...........
92
Determination of Blood ThIamine after an Oral
Test Dose of Thiamine Hydrochloride ......... ..
92
SUMMARY OF WORK WITH THE BURGH METHOD ............ ..
95
TABLE OF CONTENTS (continued)
Page
CHAPTER X
SUGGEST IONS
FUTURE WORK
BIL
A MIORO-.METHOD
. * . .
BIBLIOGRAPHY. . . . .
APPENDIX
I.
Direction
Sheet
Given to
97
100
.
106
the Subjects in Study II ...... ..
107
Table 28 - Intake of Total and. Non-Fat Oalories, Ratio
of Total to Non-Pat Calories, Thiamine Intake
y, Meg Per 1000 Oalories,
in. Terms of Meg Per
Meg per 1000 Non-Fat Calories and the Values
for thiamine in the Blood. During a 30-my
Experimental Period ......................... ..
108
III.
Directions to Subjects. ..................................
130
IV.
Calibration of Micro-Pipettes.. ........................ ..
132
Hematocrit for 5 Subjects for
52 Iys ................ .. .....................
133
II.
V.
Table 29 - Values for
LIST OF TABLES
Page
1
SUMMARY OF STUDIES IN TEE LITERATtJRE ON THE THIAMINE
CONTENT OF BLOOD.
.
.
12
2
DESCRITIONOFTHESU:BJECTS
.......
19
3
RESULTS OF TEE DETERMINATION OF STANDARD CURVES FOR
THIAMINEBYTEREEDIPEEBENTMETHODS ................... ..
31
4
TEE CONCENTRATION OF ThIAMINE IN THE BLOOD IN RELATION TO
THE MEAN INTAKE OP TOTAL AND NONPAT CALORIES, RATIO OP
TOTAL TO NONFAT CALORIES, THIAMINE INTAKE IN TERMS OF
MOG PER DAY, MOG PER 1000 CALORIES, MCG PER 1000 NONFAT
CALORIES FOR EACH GROIJP OP 5 DAYS FOR AN EXPERIMENTAL
PERIODOF3O DAYS .......................................
I ........................................ . .
StudyII ............ ... ........................... ..
Stu.dy
5
35
35
37
TEE DECREASE OF MEAN VALUES FOR THIAMINE IN THE BLOOD,
DECREASE OF THIAMINE PER 1000 CALORIES AND DECREASE IN
THIAMINE PER 1000 NONFAT CLORIES DURING STUDY I........
6
14.1
THE INCREASE OF MEAN VALUES FOR THIAMINE IN TILE BLOOD,
AND DECREASE OF THIAMINE INTAKE PER 1000 CALORIES AND
DECREASE OF THIAMINE INTAKE PER 1000 NONFAT CALORIES
7
DURING STUDY II ........................................ .
4.3
OOMPOSITIONOFBAS.ALDIET ................................
4.9
CONPOSITIONOFUNITADDEDTOBASALDIET ..................
4.9
8
COMPOSITIONOFEISCUITMIX ...............................
COMPOSITIONOFCOOKIEMIX ............................... .
50
50
9
THE DIET CONSUMED BY 5 SUBJECTS FROM JANUARY 31, 194.9,
THROUGH MARCH 21, 191+9
52
10
..
COMPARISON OF TEE THIAMINE INTAKE WITH CALORIC INTAKE FOR
5 SUBJECTS DURING THE PERIOD JANUARY 31, 194.9, THROUGH
MARCH21, 1914.9 .......................................... ..
53
11
DESCRIPTION OF THE SUBJECTS ........................... 0*
54.
12
METHOD OF DETERMINING TILE AVERAGE CORRECTED READING USED
IN THE CALCULATION OF BLOOD THIAMINE .....................
62
LIST OF TABLES (continued)
Page
13
THE pH OF THE BLOOD SUPERNA!WIT WHEN VARYING AMOUNTS OF
POTASSITJMACETATEWEREADDED.
69
1k THE pH OF THE BLOOD SUPERNATANT AFTER ADDING VARYING
AMOUNTS OF POTASSIUM ACETATE AND ACID PHOSPHATASE ...... .
70
15
16
17
DETERMINATION OF BLOOD THIAMINE WHEN VARYING AMOUNTS OF
POTASSIUM ACETATE WERE USED ............................. .
70
THE DETERMINAT ION OF BLOOD THIAMINE WHEN 2 DIFREPENT
ENZYMES WEBB ADDED TO ALIQ.UOTS OF BLOOD SUPEBEATANT EBOM
SUBJECT BWC.. ........................................... .
77
DETERMINATION OF THE CONCENTRATION OF THIAMINE IN THE
BLOOD APER 1
18
AND 2* HOURS OF INCUBATION ...............
78
THE pH OF OXIDIZED SAMPLES AF2ER THE ADDITION OP
VARYING AMOUNTS OF SODIUM DIHYDROGEN PHOSPHATE-PEROXIDE
EAGENT .................................................
19
THE CONCENTRATION OF THIAMINE IN THE BLOOD OF .5 SUBJECTS
AS DETERMINED WHEN THIOOHROI4E WAS EXTRACTED USING ISO-
BUTYLALOOHOL ............................................
20
21
81
82
THE EFRECT OF IRRADIATING THE SAMPLES OR 30 AND 60
MINUTES ............................
8k
THE AVER&GE CORRECTED READING OF SAMPLES AJER BEING
IRRADIATED ROR VARYING LENGThS OP TIME ...................
85
22 AVERAGE CORRECTED READINGS OF BLOOD SUPERNATANT PLUS
THIAMINE HYDROCHLORIDE MINUS READINGS FOR BLOOD
23
SUE'ERNA.TAMf ........................................... . . .
88
TUE AVERAGE CORRECTED READING OF BLOOD (GAS) PLUS
THIAMINE HYDROCHLORIDE MINUS TEE BEADING FOR BLOOD ..... .
90
2k AVERAGE CORRECTED READING USING THIAMINE HYDROCHLORIDE
ALONE CARRIED THROUGH TUE ENTIRE PROCEDURE ...............
25
91
AVERAGE CORRECTED BEADING USING 000ARBOXYLASE ALONE
CARRIED THROUGH THE ENTIRE PROCEDURE .................... .
91
LIST OF TABLES (continued)
Page
26 AVERAGE CORRECTED READING OF BLOOD (GAS) PLUS
COCARBOXYLASEMINUSTEEREADINGFORBLOOD ................
27
28
THE CONCENTRATION OF THIAMINE IN THE BLOOD JJER THE
ADMINISTRATION OF A 5 MG ORAL TEST DOSE OF THIAMINE
EYDROCHIOB.IE ............................................
INTAKE OF TOTAL AND NON-FAT CALORIES, RATIO OF TOTAL TO
NON-FAT CALORIES, THIAMINE INTAKE IN TERMS OF MCG PER
DAY, MOG PER 1000 CALORIES, MOG PER 1000 NON-FAT CALORIES
AND THE VALUES FOR THIAMINE IN THE BLOOD DURING A 30 DAY
EPERIMENTAL PERIOD .....................................
Study I - Nornia. .................................
Study I - Barbara .................................
StudyI-Marion ...................................
Study
Study
9.4.
108
108
110
112
111.1.
116
- Torn. .
....... . ..........................
Dave .....................................
Wesley ................................. .
Don ......................................
Keith .........................
Ruth .....................................
11.8
-
120
122
VALUES FOR EEl tATOCRIT FOR 5 SUBJECTS FOR 52 DAYS ...... ...
133
Study
Study
Study
Study
Study
Study
29
I - Roberta ....................
I - Bessie ......................
92
II
II
II
II
II
II
121.1.
126
128
LIST OF FIGURES
Page
1
2.
3,
Li..
I. *
sT.A.NDAEi O1JRVS. *
. .
MB1A1 5fl&Y THIAMNE INT.AIcE LND BLOOD THIAMINE VALUES F0R
5 SUBJECTS IN STUDY
32
39
NEAE 5DA.Y THIAMINE INTCE AM]) BLOOD THIAMINE VALUES OR
6 SUBJECTS IN STUDY II ..................................... ..
STADABDOURESUSINGTBEBU.CHNETHOD .....
......
89
THE IIETERMINATION OF THIAMINE IN THE BLOOD OF HUMAN SUBJECTS
CHAPTER I
LITERATURE
REVIEW
METHODS FOR ASSESSING THE STATE OF IDTRIT ION WITH RESPECT TO THIAMINE
According to ]nn and 1.rby (1914.5) the methods for determining the
state of thiamine nutrition may
be divided into three main classes
(1) clinical examination, (2) demonstration of biochemical
ological lesions and (3) estimation of the past
or physi
dietary intake.
Clinical Examination
Benson, Witzberger and Slobod.y (1941) used absence of anorexia,
fatigue,
constipation, calf-mu.scle tenderness and abnormal cardiac or
neurologic changes as indications of
lack of thiamine
Salcedo, Oarrasco, Jose and Valenzu.ela
incidence of beriberi in the
deficiency.
(1948) in their studies on the
Philippines made physical inspections for
signs and. symptoms of thiamine deficiency occurring in various body
regions and systems, i.e., in
the
mouth (cyanosis,
numbness
and tremor of the tongue); in the gastrointestinal
tract
of the lips
(anorexia,
vomiting, constipation and. waist tightness); in the cardiovascular
system (fatigability, palpitation, taehycard.ia, murmurs, enlarged heart
and edema); in the nervous
system (tingling,
and paresthesia); in the skin and muscles
numbness, reflex changes
(pallor,
tenderness of calf
muscles and. cramps).
Demonstration of Biochemical or Physiological Lesions
Studies on urinary excretion - The excretion of thiamine in the urine
2
of untreated subjects has been studied. in various ways: 24-hour urinary
thiamine excretions (Harris and Leang, 1936, Benson, Witzberger and.
Slobody, 191+1, Mason and. Williams, 1942, Melnicic and. Field, 1942, Keys,
HenBchel, Mickelsen and. Brozek, 191+3, Najjsr, 191+3, Perkins, 191+3,
Gifft and. Hauck, 191+6, Hathaway and Strom, 1914.6, Mickolson, Caster and.
fasting bourn excretion of thiamine in
Keys, 1914.6); the
the urine
(Najjar, 191+3, Salced.o, Carrasco, Jose and Valenzuela, 191+8) and.
thiamine excretion expressed in terms of the per cent of thiamine
intake (G.ifft and Hauck, 191+6).
The urinary response after an
or
oral
test dose of thiamine
(t1load
'satnration'1 tests) has been used. as a measure of the state of
thiamine nutrition (Melnick, Field. and Robinson, 1939,
Melnick and.
Field, 191+2, Keys, Henschel, Mickelsen and Brozek, 191+3, Perkins, 191+3,
Gifft and. Hauck, 191+6).
plete
absorption
of thiamine in
In order to eliminate such variations as incom-
of thiamine from the intestine
the
or
poasible destraction
gastrointestinal tract some investigators have pre-
ferred. to measure the urinary response to a parenteral test dose of
thiamine (Melnick, Field anti Robinson, 1939,
Mason and. Williams, 191+2,
Melnick and Field, 191+2, Alexander, Land.wehr and. Mitchell, 191+6).
Pollack, Ellenberg and. Dolger (1941) suggested. that the determi-
nation of free thiamine in the
urine
was an. index of the immediately
preceding vitamin intake only, whereas the pyrimid.ine excretion more
faithfully indicated a protracted insufficiency of thiamine.
Because
of the above possibility, the excretion of pyramine (2-methyl- 1+ amino
ethoxymethyl pyrimidino), pyrimid.ine or PAYF (pyrimid.ine in the urine
which accelerates yeast fermentation) has been measured (Pollack,
Ilger,
11enberg and Cohen, 1940, Pollack,
].lenberg and Dolger,
1941, Alexander, Land.wehr and Mitchell, 1946, Mickolaon, Caster and
lCeys, 1946).
Studies on blood or blood fractions - In an attempt to determine the
circulating thiamine in the blood some investigators
have measured the
concentration of thiamine in whole blood (Meiklejohu, 1937, Rowlands
and Wilkinson, 1938, Sinclair, 1938, 1939, Kennessy and.
Cereoed.o,
1939, Rit8ert, 1939, Widenbauer, 1939, Fiedemann and. Kmieciak, 1943,
Knott, Kleiger and
Torres-3racamonte, 19213, Oldharn, Johnston, Kleiger
and Hedderich-Arismendi, 1944, Greenberg and. Rinehart, 1945, Oldbasn,
Roberts and. Yonng, 1945, Oldbam, Ivis and Roberts, 1946).
Other investigators have studied the
in red. and white cells
concentration of
thiamine
(Good.hart and Sinclair, 1940, Gorham, Abels
and. Robbins, 191+2, Blancha.er and Cameron, 191+8, Plorijn and Smits,
1948).
Since most of the thiamine in the blood is in the form of
cocarborlase, the concentration of this phosphorylated. form of
thiamine in blood has been determined. (Goodbart and Sinclair, 1939,
Good.hart, 19/+0, Good.hart and Sinclair, 191+0).
Since thiamine is essential for the complete metabolism of pyruvic
acid and. there is a relationship between pyruvic and. lactic acid,
these intermediary metabolites
have been
determined as an indirect
means of detecting thiamine deficianey (Bu.eding and. Wortis, 191+0,
Bueding, Stein and Wortis, 1941, hied.emann and. Barborka, 1941,
Friedemann and. Haugen
1943, Horwitt, Liebert, Kreisler and Wittman,
4
1948, Horwitt and. ICreisler, 1949).
Physiological tests - Perfornce tests, such as strength, work,
muscular efficiency, endurance tests and. psychomotor responses have
been made (Keys, Hensohel, Mickolsen and. Brozek, 1943,
l'farlin, 1944).
Archdeacon and.
Since these physiological responses have some relation-
ship to thiamine nutrition they, too, are indirect means of det8rminlug the state of nutrition with respect to thiamine.
The effect of therapeutic doses of thiamine on cardiac syitoms
of thiamine deficiency has been studied. electrocardiographically
(Weiss, 1938).
Estimation of Past Dieta
Intake
Studies on the dietary intake of thiamine have been used. in the
interpretation of clinical and. biochemical data (Benson, Wltzberger and.
Slobody, 1941, 1942, Mason and Williams, 1942, Melnick and. Field, 1942,
Keys, Hensohel,
Miakelsen and Brozek, 1943, liaj jar, 1943,
Archdeacon
and. Murlin, 1944, Old.hain, Johnston, Kielger and Hed.d.erich-Arisrnendi,
19)44, OlcIham, Roberts and Young,
Gifft and. Hauck,
1945,
1946, Hathaway and
Alexander and. Lsndwehr, 1946,
Strom, 1946, Mickelsen, Caster and
Keys, 1946, and Oldham, Ivis and Roberts,
1946).
The relationship of thiamine to total Calories and. of thiamine to
non-fat Calories have been used as other measures to determine the
adequacy
of the thiamine intake (Oowgill,
1934,
Williams and. Spies,
1938).
It seems to be the general opinion that no one test alone is
sufficient to determine thiamine deficiency
or sufficiency.
The
consideration of the relationship of one test to another and. of the
conditions under which the tests are performed is of necessity a
Mason and. Williams (191+2) stress the need of
requirement.
intelligent
interpretation of these various indices.
THODS OR TIlE ITERMINAT ION OP INTERI.tEDIARY METABOLITES, THIAMI1E A1
000ABBOXYLA.SE IN WHOLE BLOOD OR BLOOD PRACTIONS
Pyru.vic and lactic Acids
Thiamine pyrophosphate, or cocarboxylase, is the coenzyme
necessary for
the
normal catabolism of pyruvic acid in the body tissues.
Since there is an accumulation of pyruvic acid. in the blood in cases of
thiamine deficiency, Bu.eding and. Wortis (191+0) proposed a method for
the determination of blood pyrvate which is a modification of the
method of Lu (1939).
In order to emphasize the presence of the deficient state, some
workers have added a 'metabo1ic loads such as exercise.
and blood pyruvate both increase after exercise.
Blood lactate
iedemann and
Barborlca (191+1) found that there was a definite ratio between lactic
acid. and pyruvic acid in the blood.
proposed. a method for
Barker and Swnmereon (191+1) have
the determination of lactic acid and. Friedom-nn
and Haugen (191+3) have proposed a method. for the determination of keto
acids in the blood.
Baeding, Stein and Wortis (191+1) increased. the metabolic load by
giving their subjects an oral dose of glucose.
They determined. the
pyruvic acid and. the glucose of the blood from subjects in the fasting
state and repeated their determinations at intervals after the test
dose of glucose was given.
They found that in deficient subjects there
was a prolonged elevation of the pyruvic acid. content of the blood and.
a slightly elevated blood sugar content when compared with values for
normal subjects.
Ho
itt and. Kreisler (19k9) and. Horwitt, Liebert, Kreisler and
wittman (l9Li.8) found that in the fasting state and. in the absence of
exercise there was no consistent rise in the basal levels of lactic
acid. and pyruvic acid in mild. degrees of thiamine deficiency.
They
combined glucose ingestion and. mild exercise to form a double meta-
bolic load. thus providing a still greater strain on the mechanism of
carbohydrate metabolism.
The authors suggested that early thiamine
deficiency could. be diagnosed if the glucose level of the blood was
correlated with the lactate and pyruvate levels of the blood when both
were determined under the double metabolic load, of glucose ingestion
and exorcise.
The difficulty with these methods is that so many factors
influence the concentration of pyruvic acid, in the blood.
The concen-
tration of pyruvic acid depends greatly on the degree of physical
activity which makes difficult the interpretation of slight changes in
the pyruvic acid. content of the blood (Nutrition Reviews, l98).
Thiamine in
asma and Serum Compared with that of Whole Blood
Meiklejobn (1937) found that whole blood has greater growthpromoting activity for Phycomyces blakesleeanus than did plasma.
He
found that about 20 per cent of the total thiamine was present in the
plasma but Good.hart and. Sinclair (1939) reported that this value may
be high since any hemolysis which might have occurred. would increase
greatly the value obtained for the thiamine content of plasma.
irthermore, Gorham, .Abels and Bobbins (l9L2) found that the plasma of
a group of normal individuals was substantially free of thiamine.
Goodhart and. Sinclair (1939) reported that 90 per cent of the thiamine
in the blood is in the form of cocarboxylase and. that all of the blood.
cocarboxylase is contained in the blood cells.
Benson, Witzberger,
Slobody and. Lewis (191+2) stated. that only about 10 per cent of the
total blood thiamine is present in the plasma and that this is present
as free thiamine.
These studies would indicate that very little thia-
mine, either free or combined is present in the serum or plasma.
For
this reason determinations on these blood fractions would not be a
satisfactory index of the amount of thiamine in the blood.
Therefore,
determinations for total thiamine, i.e., cocarboxylase plus free thiamine, must be carried out on whole blood rather than plasma or serum.
Thiamine in the Formed Elements of the Bloo4
Goodhart and Sinclair (191+0) indicated. that a mach larger amount of
cocarboxylase was found in mrnlian leucocytes than in erythrocytes.
Gorbam, Abels and. Bobbins (191+2) adapted the micro-tecbnique of
Atkin, Schultz and Frey (1939) and. found in the white blood cells of 30
normal adults a total thiamine concentration of 1+8 to 183 meg per 100 ml
or an average of 99.8 meg per 100 ml.
The total thiamine in the erythro-
cytes of 21+ of the 30 normal adults was 3.7 to 38.0 meg per 100 ml or an.
average of 10.3 meg per 100 ml.
The average thiamine level for normal
white cells was therefore about ten times the amount contained in
normal erythrocytes.
The authors stated that this was similar to
E1
observations on the distribution of riboflavin and ascorbic acid in
blood cells.
They suggested that the greater concentration of
thiamine, riboflavin and ascorbic acid in the white cells may be
explained by the fact that of the several blood components, the white
cells most closely resemble actively metabolizing tissue.
Blanchaer and Cameron in 19148 using the Lactobacillus fermentim
method (Sarett and Cheldelin, 19144, Cheldelin, Bennett and. ICornberg,
19146) found 10.8 to 27.6 meg of thiamine per 100 ml of blood cells in
23 patients who exhibited a wide range of nutritional status.
!lorijn and. Smits (19148) determined the thiamine pyrophosphate
content of red and white corpuscles.
They found that the average
thiamine pyrophosphate content of red cells of 8 normal men was
1.149± 0.08 meg per 1011 cells and, of 114 normal women, was 1.28
meg per 1011 cells.
,
0.09
The average thiamine pyrophosphate content of
white cells was 280
145 meg per 1011 cells.
Thus, for human beings,
Tlorijn and Smits found the ratio of the cooarbolase content of a
red. to a white cell was about 1:200.
The sex difference was significant
for red. cells but not for white cells.
Cocarboxylase in Whole
iood
The cocarboxylase method (GQodbart and Sinclair, 1939, Goodhart,
19140) is based on the fact that thiamine markedly stimulates the
decarboxylation of pyruvic acid, by alkaline washed yeast in the
presence of cocarboxylase.
in Warburg manometers.
The evolution of carbon dioxide is measured
Since nearly 90 per cent of the thiamine in the
blood is in the phosphorylated. form (Goodhart and Sinclair, 19140) this
method estimates the major portion but not the total amount of thiamine
I.J
in the blood.
Thiamine in Whole Blood
Phycomyces blakesleean'as - Meiklejohn (1937) suggested the use of a
mold, Phycomyces blakesleeanus, as a test ornism for the determination
of total thiamine since small amounts of thiamine have a growthpromoting activity on this organism.
Rowlands and Wilkinson (1938) and
Sinclair (1938, 1939) have also used. this method with modifications.
It is assumed that the growth of the mycelium obtained with blood.
represents the true maximum growth that the thiamine present in the
blood. can effect.
Sinclair (1938) criticized the method. of Meiklejolrn
since the method. is not specific for thiamine.
Both thiamine and its
breakdown products aid. in the growth of Phycomyces blakesleeanus when
both of the products are supplied together.
Fermentation - In 1937 (a, b), Schultz, Atkin and Frey noted that
thiamine exerts a "powerful action" on the rate of alcoholic fermentation.
This rate of fermentation is expressed by the amount of gas
evolved in a given time interval.
In 1939, Atkin, Schultz and
ey
modified the test for use with a Warburg apparatus in an atmosphere of
nitrogen.
The fermentation stimulation is not a specific test for
thiamine since 2 - methyl - 5 ethoxymethy3. - 6 aminopyrimidine is also
effective in stimulating fermentation (the thiazole group is inactive).
The authors suggested that the results be correlated with the animal
growth method to check the accuracy of the indirect method for the
determination of thiamine in blood..
Knott, Kleiger and Torres-Bracamonte (l9J43) suggested the use of
10
the modified Atkin, Schultz and Prey method in the determination of
blood thiamine0
This method was used by Oldham and coworkers in three
studies (1) Oldham, Johnston, Kielger and Hed.dericb-Arismendi (19)44)
in a study on two 5-year old. boys, (2) Oldbam, Roberts and. Young (194.5)
on children 6 to 15 years of age and (3) Old.ham, 1vis and. Roberts
(194.6) in a study on young women 19 to 32 years of age.
Thlochrome - The thioclirorne method for the determination of thiamine is
a chemical procedure and depends on the oxidation of an
alline
solution of thiamine by potassium ferricyanide which converts the
thiamine present to thiochrome, a yellow substance having an intense
blue fluorescence.
The thiochrome is extracted with isobutyl alcohol
which separates it from any physical interfering substances occurring
in the sample or from the reagents used in the analysis.
fluorescence is
considerably brighter in
Thiochrome
isobutanol than in
water so
this adds to the sensitivity of the method (Hennessy, 194.7).
blood
Ritsert (1939) suggested a thiocbrome method for
after
precipitation of the protein with saturated sodium sulfate at 80 to
90 degrees Centigrade.
free thiamine
He concluded that in human blood there was only
and. no pyrophosphate
present.
Wideubauor (1939)
suggested that Ritsert precipitated the unsplit cocarboxylase - protein
comulex with sodium sulfate and this accounted for the fact that the
discarded water medium showed no blue fluorescence.
Wid.enbau.er
suggested the splitting of the cocarboxylase-protein complex by heating
serum, water and hydrochloric acid. in a hot water bath for 10 minutes.
Honnessy and Cereced.o (1939) suggested a method for the estimation
1].
of free and. phosphorylated thiamine by the thiocbrome method.
They
suggested enzymatic hydrolysis to obtain all of the thiamine in the
free form and the use of base exchange to replace the adsorption
technique that had. been used. previously to separate the thiamine from
other constituents contained in the medium.
(19ii-3). published a
(l9L5)
Kmiociak
method using five ml of blood, that was a
simplification over previous
Rinehart
Friedemaun and
thiochrome
methods and Greenberg and
suggested a further simplification of the thiociromo
method. using only one ml of blood.
The phosphorylated. thiamine is not measured satisfactorily by the
thiochrome method. without preliminary enzymatic hydrolysis.
There are
fluorescing substances other than thiochrome in biologic material
which result in some lack of specificity but Youmaus and. Patton (l9Al2)
stated that under ordinary conditions this is not a serious objection
to the method.
However, they also stated that the method. requires
considerable technical care to be used satisfactorily.
Ranges of values for the thiamine content 0
whole blood. as
determined by the various methods are: Phycomyces blakeseeanus, 6.5 to
16.5 mcg per cent; Fermentation, 2.6 to 7.7 meg per cent; Thiocirome,
2.8 to 15 mog per cent (Table 1).
In both the fermentation and.
thiochrome methods the lowest values, 2.6 and 2.8 meg per cent are
associated with lowered thiamine intake.
12
Table 1
SIJIvINA.RY OF STUDIES Ii
-
T1
LITERATURE OI THE T1II.AM1EE C0I'ITENT CF BLOOD
Thiamhie Inta1
-
Goodhart and Sinclair
1939
cocerboxylase
Goodhart
cocrboxylase
Rlood Thiamine
Mean
Range
rncJl0OOQ
26 men
mc
mc
7.0+2.1
4.5-10
7.0±1.53 S.D.
(2o-Mo yrs)
28 boys
(5-is
1940
yrs)
Powlands and Wilkinson
1938
Phycomyces
adults
Sinclair
1939
Phycomyces
144 observations
on author
6.5-16.5
over period of 3 yrs,
various conditions
45 adults (both
8.0-13.5
9.5±1.5
5.5-10.5
7.l4-l.LL
sexes)
adults
Goodhart and Nitzberg
1941
microfermentation
14.5
Knott, Kleiger, TorresBracamonte
microfermentation
37 lactating
women
3.1- 9.2
fasting blood
bloods taken 8 or
lO:3O not fasting
5.39
5.01
1 9143
6.i6
15 non-lectating
women
(caic)
OldMm, Johnston,
Kleiger and
Heddrich-Arismendi
microfermentation
2 boys
(5 yrs old)
fasting blood, 3 months
study, 36 periods of
3 days each
19414-
225,250
475,500
39 children (Oct)
children (May)
(6-15 yrs old)
4-0
fasting blood
3.6
575,575
750,750
775,825
900,875
8.4, 7.1
7.5, 7.7
800,800
microfermentation
4.9,
6.9, 4.8
6.3, 5.3
4-800,48oO
OldEam, Roberts and
Young
1945
S.D.
l4..5._12.O
-
-
-
8.4, 6.5
5.c
7.3
13
Table 1 (continued)
J
Blood Thiamine
Range
Mean
Authors
Oldham, Davis
and Roberts
1946
microfermentation
12 women
(19-32 yrs)
8 months stidy, Blood
tsken at end
of each period
preliminary (7 days)
samples
-
59 days
47 days
-
295
424
140
200
days
742
937
4900
4914
1027
45
41 days
7 days
7 days
20 days
4.0-6.7
5.2
3.8
360
510
2.6-5.0
3.7-6.7
6.3-7.2
4.8-6.4
540
5.5-6.7
6.2
-
-
E!ennessy and Cerecedo
1939
thiochrome
9
-12
Ritsert
1939
thiochrome
3
-15
Benson, Witzberger,
thiochrome
Friedemann and Kmieciak
thiochrome
1943
GreenberE and Rinehart
1945
45 children
(L_12
Slobod.y, Lewis
1942
thiochrome
yrs)
fasting blood 1 week
cal. ad. lib (1624-3051)
7 women
29 men
not fasting blood
not fasting blood
BJ
3 weeks study
high calorie diet
beginning
2d & 3d week
TF
3 month (3 detns)
TF
2 weeks
630-1170
4.8-12.3
3.0-9.2
3.8-11.2
5.3
6.8
5.6
-
-
7.81.3
5.60
5.73
1500-1900
6.1
2500
300
4.3-5.5
4.7
7.0,9.9,9.
8.9
2.8
5
-10
-
'4
T
CTJESTI0N OF THB SIGNIFICANCE OF BLOOD THIAMINE VALUES IN ASSESSING
TNE STATE OF TJTRITION WITH BESPEOT TO THAT VITAMIN
There are many differences of opinion regarding the value of the
determination of thiamine in the blood as an indication of nutritional
status with respect to that vitamin.
Goodhart and Nitzberg (1941) found in their subjects whose dietary
intake
ranged. from 898 to 1493
could be found
vitamin to
between
meg of thiamine daily that no correlation
urinary
values.
the
thiamine intake,
Calorie ratio of the diet or fasting
However, they found that there was a
peripheral
excretion1
definite
the
blood. levelsof thiamine.
association between acute
neuropatby of the alcohol addict and. low blood thiamine
The incidence of peripheral neuropathy among subjects with low
blood thiamine values was so high that the authors stated. that it
would seem likely that further work would prove a blood thiamine value
below 3.0 meg per cent, by the fermentation method, a
indication of a thiamine deficiency
definite
state.
Benson, Witzberger, Slobody and Lewis (1942) carried out a study
on 45 hospitalized children who were clinically
healthy and known to be
in a state of thiamine saturation.
Four pre-breakfast blood determi-
nations were made during one week.
They found that the daily variation
in blood thiamine of an individual child did
not follow the urinary
thiamine output and that it was not proportional to the amount of
thiamine excreted or to the per cent of dietary thiamine excreted. in
the
urine.
They suggested that there may be an individual fixed blood
thiamine concentration in a thiamine saturated individual which cannot
15
be raised..
later, in 1943, Benson,
Witzberger and Slobody compared
the
above study with blood values obtained on 121 children who were
hospitalized, 22 of whom showed iinquestionable evidence of tissue
unsaturat ion.
cally healthy
They found similar blood values for both the 4,5 cliniThey
children and the 12]. children in the later study.
concluded that the concentration of
thiamine
in the blood is not
indicative of the state of nutrition with respect to that vitamin.
Youmans and. Patton
(1942) stated that the value of blood thiamine
determination in the diagnosis of thiamine
deficiency seems to be
limited to instances of severe clinical deficiency, e.g., peripheral
neuritis.
They stated that their investigations indicated that the
thiamine content of
the
course of a deficiency.
blood. does not decrease until late in the
Lto to these observations they felt that the
"estimation of the thiamin level in the blood does not serve as a
satisfactory index of thiamin deficiency previous to the onset of
outspoken clinical evidence of the disease".
Oldham, Johnston, Xleiger and Heddericb-.Arismendi (1944) found in
studies on two 5-year old boys that the correlation coefficients of
blood. thiamine with fasting one hour excretion, with 4-hour, with
24-hour return of a test dose and with average intakes were
0.58, 0.40 and 0.45, respectively.
Old.ham et a]..
concluded
0.66,
that
blood thiamine seems less reliable than excretion tests as a measure
of nutritional status.
Hennessy and Cerecedo (1939) stated that they found the concentration of thiamine in the blood and in the urine to be of the same
magnitude.
16
Knott, Kleiger and Torres-Bracamonte (l913) analyzed Ui samples
of milk from 50 women and also determined the blood thiamine of 37 of
these women.
They found a definite tendency for low milk values to
be associated with low blood thiamine values and higher milk thiamine
with higher blood thiamine values.
Friedemann and Kmieciaic (191+3) stated that the total thiamine
content of blood does not fluctuate as widely as urinary thiamine
excretion and that it is not as rapidly affected by
of thiamine.
the
dietary intake
However, they felt that there was a very definite
correlation between the concentration of total thiaminein the blood
and. the incidence of deficiency symptoms.
One subject on an intake of
approximately 2500 meg of thiamine per day had. values of 7.0, 9.9 and
9.9 meg per cent of thiamine in. the blood when determinations were made
at monthly intervals.
After two weeks during which the subject received
a diet containing about 300 meg of thiamine per day the blood level
fell to 2.8 meg per cent.
In most of these studies determinations of blood thiamine were
made on only a few subjects, and, in general, the concentration of
thiamine in the blood was followed for only a short period of time.
some investigations,
the fasting state.
In
the blood samples were not drawn from subjects in
Because of the differences of opinion regarding
the significance of blood, thiamine values it seemed of value to carry
out a controlled study on the thiamine content of the blood of human
subjects: (1) when the subjects were allowed thiamine-containing foods
17
ad. 1ibitui and. the food. values calculated. for that period and (2) when
subjects were maintained on a controlled diet, supplemented by two
levels of thiamine for 3 weeks each,
18
OE&PTER II
PRO CEDtJPE FOR THE EXPERIMENTAL STUDY IN WHICH THE
IEDEMA.NN
KMIECIAX METHOD WAS USED FOR TEE DETERMINATION Q ELOOD THIAMINE
PLAN OF EXPERIMENTS
Study I was conducted. on. 5 students from October 22 to November
and Study II was conducted on
16,
6
students from
January 31 to
February 29, 19L8.
The subjects wore on 30day studies in. which their intake of
ascorbic acid only was
controlled.
consecutive 10day periods.
During period 1 the
]iitum at meals and a record was
all foods
ascorbic acid each day in order to
vitamin C.
containing
kept of each subject's
significant amounts of ascorbic acid,
supplement
During period 3
recommended allowance
2 the
of crystalline ascorbic acid so that
equal to the recommended allowance
Research Council.
to 20 mg
This of necessity limited or
fruits, cabbage and potatoes. During period
were given a
intake was
than the
allowed
achieve saturation with respect to
of ascorbic acid in their food.
such as citrus
the
were
During periods 2 and 3 the subjects were restricted
eliminated all foods
subjects
subjects
Daring this period the subjects were also given 200 rng of
food intake.
or less
The study was divided into three
the
of the
of the
National
subjects were given 10 mg less
National Research Council for
ascorbic acid.
Study I was conducted at
Snell Hall, a girl's dormitory
on the
Campus near the Home Economics building. A special table was assigned
19
Before each meal the research workers
for the experimental subjects.
The hot foods were weighed out and
weighed out all the cold foods.
served when the subjects arrived.
Study II was conducted at the Memorial Union Cafeteria.
The same
procedure of weighing and serving foods was carried out there also.
SCBIPT ION OF SUBJECTS
The subjects were all
presumably
normal,
derately active students who were
healthy individuals.
Eight of the students were
18-year old Freshmen and. three were adults.
A
description of the
subjects in terms of age, height, weight at the beginning and end of
the study, weight range and average weight is given in Table 2.
Table 2
DESCRIPTION OP THE SUBJECTS
Body Weight
Beg.
of
Study
lb
End
of
A1e
yr
Heibt
Norma
Barbara
Marion
Roberta
Bessie
18
18
18
18
27
65 1/2
65
65 1/2
117
116
117
111.9
1114 1/LI.
6L4. 1/2
156 1/2
110
151
Tom
ve
Wesley
Dou
Keith
18
18
18
73 1/2
73 1/2
1814.
66
18
70 1/2
126
150
27
30
67
1113
624.
153 1/2
Subject
Ru.th
in
62 1/2
Study
lb
1/11.
Average
Bane
Wei,g
lb
lb
117
116
109 3/24'
115 1/4-118 1/14.
117 1/2
ll4
1149
1411.
-156 1/2
151
108 3/11-lu
186
183
183
178
186
182
128 1/2
126 1/4-131
153
1116 3/24*153
3/14.
151
1115
1111 l/2_1L1.5
1/2
11421.
124.2 1/2
121.1
1/2-153
1/14.
116
1111. 1/2
178 1/2
Weight
1/14.
-189
185
1115
153
110
129
20
GEIRAL DAiLY PRO OEDTJEi
The subjects were given a general direction sheet so they could
acquaint themselves with the procedure and what was expected of them.
All subjects were very cooperative in eating all the food served and
adhering to the necessary restrictions imposed upon them.
A copy of
the directions given in Study II is found in. Appendix I.
The daily procedure was the same throughout the study.
The
subjects reported to the nutrition research laboratory at 6:Li5 A.M.
They weighed themselves (without shoes) daily and recorded. their weights
Five
on a record sheet posted near the scales inside the laboratory.
ml of blood were taken by vonipuncture every 5th day for the determination of the amount of thiamine in the blood.
The subjects reported
to Snell Hail (Study I) or to the Memorial Union (Study II) at meal
times when all the foods were weighed out for them.
spring balance was used. for weighing the foods.
the food intake of each of the subjects.
as possible.
A Ohatillon
A record was kept of
Mixtures were avoided as much
Pits or rin.&s were removed from fruit.
Bone and. gristle,
and some fat if necessary, were removed from the meat so that the
edible portion only was weighed.
Eggs were weighed in the shell and
then the shells were weighed back and. subtracted to obtain the weight
of the edible portion.
Values were calculated for protein, fat,
carbohydrate and. thiamine content of the foods.
21
CRAPTER III
KMIEOIAX 1ETH0D
ANALYTICAL PROCEDURE FOR THE FRIEDEMA11I(
EQUIPMENT USED
The method. of Fried.omann and. Kmieciak (].9M.3) was followed. for the
determination of thiamine in. the blood.
The eauipment used for this
procedure was as follows:
1.
5 ml hypodermic 8yringee
2.
20 and 22 gauge
3.
Oxalated. tubes prepared by adding by burette 0.1 ml of 2%
lithium oxalate
hypodermic
for
needles
each ml of blood taken.
dried aro'und. 60 degrees Centigrade.
The tubes were
Too high a temperature
dioxide from the oxalate.
will drive off carbon
Non-protein-nitrogen tubes (graduated at 25 and 50 ml)
5.
Footed stirring rods -
long rods
were made with a 0penny"
shaped end. that fitted the bottom of the
type of rod. the contents of
the
PN tube.
With this
tube were agitated violently
by rapidly pulling the rod. up and. down in the 1N tube.
6.
Boiling water bath
7.
Incubator
8.
Large centrifuge and cups
9.
Centrifuge tubes -
heavy-walled, 90 ml and. 50 ml capacity
10.
Erlenmeyer flasks - 150 ml capacity
11.
Stop watch
12.
Set-up for suction
22
13.
Oapil1ay tube for use with suction
11+.
Coleman photofluorometer
15.
Optically matched. cuvottes for the Coleman photofluorometer
16.
Hennessy adsorption columns
17.
Thiamine reaction vessels (Wilkens-Anderson Co., Chicago)
50 ml capacity
18.
Syringe pipettes to hold. 10 ml and 20 ml hypodermic syringes
(Northern Tool and Instrument 00., Flushing, N. L)
19.
Automatic timer
20.
Volumetric pipottes - 2,
21.
Graduated pipettes - 2, 5, 10, 25 ml
22.
25 ml graduated
5, 10 ml
cylinders, with, glass stoppers.
REAGEITS USED
The reagents used, in the Fried.emann and Kmieciak method. were the
following:
H01
1.
Approximately 1.0
2.
Approximately 1.0 4 NaH003
*3
10% metapbosphoric acid, prepared fresh each day.
in cold water.
*Lf.
Disso1ed
kept In refrigerator.
Phosphatase - io% Clarase 300, prepared. daily.
If the
clarase thus prepared was not clear, it was either filtered.
or centrifuged..
5.
Ion exchange adsorbent - 1 kilogram of Decalso (artificial
zeolite) was covered. and. mixed with four changes of acidified.
* Reagents
prepared daily
23
solution of NaOl.
(2 ml concentrated EC1 per liter) 25
When
ordinary table salt was used in Study I the solution was
filtered bofore the addition of acid.
removed by decantation each time.
The supernatant was
The zeolite was washed five
or more times by decantation with acidified distilled water
(1 ml glacial acetic acid per liter).
Washing removed the
finer material as well as the excess salt.
The Decalso was
then transferred to a large Buchner funnel and washed well with
distilled water and dried at room temperature in. a shallow pan.
To have the Decalso in the maximum expanded state a small
portion of the activated Decalso was kept motstened in a wide
mouth bottle.
6.
Sodium chloride solution - 250 grams of 0. P. NaCl plus 2 ml
of concentrated HOl were diseolved and diluted to one liter.
According to
riedomann and Kxnieciak (l9L3), salt solutions
which are prepared from ordinary table salt, contain materials
which slightly reduce the yield. of thioohrome.
When table salt
was used. (in Study I) the solution was filtered before
acidification with HOl.
*7.
Oxidizing reagent - mixture of nine parts of 10
part of 1% potassIum ferricyanide.
HaOH and. one
This mixture was freshly
prepared each day.
8.
Iso-btityl alcohol - with a fluorescence no greater than
distilled. water.
If the alcohol contained. much fluorescing
material it was acidified with concentrated HC1, dehydrated
*
Reagents prepared daily
2J4
The distillate
with anbydrous sodium sulfate end distilled.
which came over between 106 to 108 degrees Centigrade was
collected and. used. in the extraction of thiochrome.
Used
alcohol was poured into a bottle containing excess HOl and.
sodium sulfate and this was later rediatilled.
All new iso-
butyl alcohol was redistilled. collecting only the 106 to 108
degree boiling fraction.
9.
quinine sulfate stock standard - 100 mg quinine sulfate,
U.S.P., made up to one liter with approximately 0.1
acid.
bottle.
This was stored in the refrigerator in an amber-colored.
A solution kept in this manner should be stable for
at least one year (Friedemaim and Kmieciak,
10.
sulfuric
l9i4.3).
quinine sulfate intermediate standard. - 25 ml stock standard
diluted to 100 ml with approximately 0.1
sulfuric acid.
This was stored in the refrigerator in an amber-colored
bottle.
The intermediate standard was made up new about once
a week although it probably would. have been stable over a
considerably longer period of time.
*11.
quinine sulfate working standard - exactly 5 ml of the intermediate standard were diluted to 500 ml with approximately
0.1 N sulfuric acid.
This solution was prepared each day.
Until used the working standard. was protected from the light
by storing in the desk in the dark.
12.
Thiamine stock standard - exactly 50 mg of anhydrous thiamine
hydrochloride (dried several weeks over sulfuric acid in a
*
Reagents prepared. daily
25
desiccator) were dissolved in 25 per cent ethyl alcohol in
approximately 0.01! H01.
When brought to a voimme of 500 ml
with this solution each ml contained 100 meg of thiamine
hydrochloride.
If kept cold thia stock solution should be
stable at least six months (Fried.emann. and Kmieoiak, l93).
Thiamine intermediate standard - 2.5 ml of stock solution
13.
were made up to a volume of 250 ml with approximately 0.01
N HOl.
One ml contained 10 meg of thiamine hydrochloride
Thiamine working standard. - 5 ml of the intermediate solution
were diluted to 250 ml with approximately 0.01
ml of the working standard
contained 0.2 meg of
HOl.
One
thiamine
hydrochloride.
Indicator - brom phenol blue.
15.
Five drops of a 0.L4. per cent
solution were used.
Os.prylic alcohol.
16.
TERNIWION OF THIAMI1B IN TI
BLOOD
Collection of Blood Sa1e
Five ml of blood were collected by venipucture in. a hypodermic
The blood was delivered into an oxalated tube and. shaken
syringe.
vigorously until the dark red venous blood. bad. changed to a bright red
color.
One drop
of eaprylic alcohol was
added to reduce foaming.
Since the thiamine content of the blood gradually changes even in the
cold, it is necessary to perform the analyses without delay.
For this
reason, the thiamine analyses were begun as soon as the samples were
measured.
Peciitation
Digestion with Phophatase
1.
xactly
t
Protein
5 ml of blood were delivered into a
large heavy-
walled test tube (1'N tube) containing 25 ml of distilled
easier
water (if the blood was cooled slightly it was
pipette since
fewer bubbles were
produced in
to
pipetting).
The 5 ml volumetric pipette was rinsed by drawing up the
waterblood mixture and blowing it out into the tube.
This
was repeated five times.
2.
The contents
Two ml of approximately 1.0 ! HO]. were added.
of the tube were mixed with a footed glass rod.
kept in the tube rntil
motaphosphoric acid
3.
the
The rod was
protein was precipitated with
as described in paragraph 8.
The tubes were heated 10 minutes in a boiling water bath and
the contents stirred frequently.
Lj.
The tubes were cooled by placing in a pan of cold. water.
drop of caprylic alcohol was added.
One
1 to 2 ml of approximately
1.0 j NaBCO3 was added slowly with stirring.
The volume of
the NaHCO3 must be sufficient to bring the pH between 14.5 and
5.5.
In this study 1.3 ml were used to produce a pH close
to 5.0.
5.
exactly 2 ml of the clarase solution were added.
6.
The tubes
were then kept 1.5 honre in a water bath at 140 to £5
degrees Centigrade.
by stirring.
We
The contents were mixed every 15 minutes
placed the tubes in a pan of water which was
between kO to 145 degrees Centigrade and put the pan in an
incubator to maintain the tubes at that temperature
range.
The temperature was checked each time the tubes were mixed
to be sure the incubation temperature was being maintained.
7.
The blanks
were prepared by
order indicated.
adding the same reagents in the
These were incubated and. carried through
exactly the same procedure as the blood
8.
samples.
Ten ml of io% nietaphosphoric acid were added with a syringe
pipette.
The volume was adjusted to 50 ml with distilled
water and the contents were mixed.
This
mixture was poured
into a centrifuge tube and centrifuged 15 minutes.
supernatant was poured off into a 150 ml
The
rlenmoyer flask.
Since, according to Friedemann and Kmieciak (19)43), the
precipitate may contain some adsorbed thiamine which may be
recovered by re-extraction, 2.5 ml of metaphosphoric acid
were added to the
N tubes and the volume again brought to
50 ml with distilled water.
The contents of the tube were
transferred to the centrifuge tube containing the precipitate
and mixed well.
This process rinsed the NPN tube.
The rod
was rinsed with distilled water and the washings allowed to
flow into the centrifuge tube.
The tube was centrifuged again
for 15 minutes and this supornatant was added to the same
Erlenmeyer flask so that the final volume was about 100 ml.
9.
five drops of brom phenol blue were added to the Jrlenmeyer
flask containing the combined extracts.
adjusted to pH 3.0 to 3.6 with 1.0
The contents were
NaHCO3.
To check on. the
colcr and pH, the pH of one flask was checked with the Becan
pH meter and then all the other samples adjusted to that
color.
At this point the solutions were stored in the
refrigerator overnight.
Adsorption and. Elutio
10.
The solutions were removed from the refrigerator and the
color noted to be sure that there had been no change in pH.
11.
Preparation of adsorption columns - these were made up ahead
of time, usually the night before, and kept in a beaker of
distilled water to cover until ready for use.
A plug of
glass wool was pushed into the bottom of a Honnessy tube.
The tube was filled with distilled water and the Decalso
added, a small amount at a time, while the tube was held at
a slight angle.
Air bubbles were avoided.
Decalso was
added until an excess, forming a layer several mm deep, was
present in the bell above the column.
The adsorbent is more
uniformly packed when floated into place this way.
The excess
Decalso in the bell prevents clogging of the column by solid
or colloidal material.
This also prevents access of air into
the column provided the drained column is not jarred
(Friedemann and Kmieciak, l9Ll3).
Just before ready to use,
the adsorption column was placed in a holder (spring typo
broom holders were used) fastened on a shelf above the desk
and allowed to drain.
The extract was passed through the
adsorption column.
12.
The 150 ml Erlenmeyer flask was rinsed. 3 times with 10 ml
portions of distilled water and the washings passed. through
the adsorption column.
The column was allowed to drain
29
completely after each washing.
The bell of the column was
then washed once with 5 ml of distilled water and the column
again allowed to drain completely.
13.
Three ml
portions of
the acidified NaOl solution were added
until exactly 25 ml were eluted into the 25 ml graduated
cylinders.
Oxidation to Thiochrome
Two 10 ml aliqu.ots of the eluate were transferred to thiamine
reaction vessels of 50 ml capacity.
15.
Five ml of the oxidation mixture were added with a syringe
pipette.
Thia oxidation mixture was added rapidly to produce
mixing.
16.
Thirteen ml of iso-butyl alcohol were added immediately with
a syringe pipette.
17.
Thiamine reaction vessels were stoppered
and shaken vigorously
for 90 seconds.
18.
The reaction vessel was centrifuged 2 minutes at slow speed
to separate the layers.
The lower layer was drawn off by
means of a capillary tube attached to suction.
3y passing
the capillary tube rapidly through the upper layer the lower
layer was easily removed with negligible loss of the iso-butyl
alcohol layer.
19.
Three to
four 0.2
were added.
solution
gram samples of anhydrous sodium sulfate
The first addition entrapped the remaining
at the bottom and the addition of more sodium sulfate
removed the water from the alcohol phase.
The tube was shaken
30
gently and centrifuged 1 minute.
If the alcohol layer was not
crystal clear more sodium sulfate was added and the reaction
vessel recentrifuged.
20.
The clear iso-butyl alcohol layer was poured into a matched
optically clean cuvette.
The fluorescence was measured in
the Coleman photofluorometer against the quinine sulfate
working standard set at 70.
21.
The thiamine content was determined from a standard curve
representing the increase in fluorescence after the addition
of 0.2,
0.6, 0.8 and. 1.0 meg of thiamine hydrochloride
to blood samples which were analyzed by this procedure.
That
is, to 5 ml blood samples were added 0, 0,2, 0./+, 0.6, 0.8 or
1.0 mog of thiamine hydrochloride.
These were carried through
the procedure after which the value for blood thiamine only
was subtracted to give the fluorescence due to the added
thiamine.
A new standard curve was set up for each study just
before or during the course of the study.
DTBMflATI01 OP STJDABD CURY
During the preliminary work standard curves were determined by
3 different methods:
1.
Pure thiamine solutions were carried through the oxid&tion
procedure only.
2.
Pure thiamine plus blood minus the thiamine of the blood alone:
In this method, 25 to 30 ml. of blood were collected and known
31
amounts of standard thiamine solution were added to 5 ml
samples of blood.
method.
These were carried through the entire
The reading for blood thiamine alone was subtracted.
from the reading of blood plus thiamine to give standard.
(This curve was the one finally used in
curve figures.
calculating blood thiamine).
3.
Pure thiamine treated as blood:
carried through the entire
The sample
Figure 1.
Pure thiamine solutions were
procedure.
readings minus blenk readings are given in Table 3 and.
There was a
than
greater similarity between curves 2 and 3
between curves 2 and 1 since the entire procedure was not followed to
obtain the results for curve 1.
Table 3
RESULTS OP TH DETEBMINAT ION OF STARflARD OURVES
FOP. THIAMINE BY THREE DIFFERENT METHODS
Sample reading - blazk reading
2
3
Pure thiamine
Pure thiamine
Pure thiamine
treated the same
+ blood - blood
as blood
thiamine
1
Mcg thiamine
0.2
J4..75
6.09
10.75
10.78
9.50
0.6
18.00
16.50
lLf.50
0.8
25.25
20.60
19.00
1.0
3A4..0O
2.lO
2L.13
32
STANDARD CURVES
FIGURE 1.
£1
(0
h
F
I
'I
II
1
II
a,
'I
II
'I
I
0.1
/
/
II
07
/
/
/
1
/ /
II
Ii
II
-..1
''
,/
II
I,
/
,
/
/
/
4j
Qs
CVRVE 1:
/ I
1,7
THIAP/INE
HYDROCHLORIDE
/ 'I
C(/RVE 2:
1,'!
(THIAMiNE
HYDROCHLORIDE +
BL 000) -BL 000 THIA Il/N E
/7
0.2
-----C(IRVE3: THIAMINE
HYOROCHLOR IDE
TREATED AS BLOOD
01
00
0
5
10
15
20
25
30
O8SERVED READING - BLANK READ/N&
33
In calculating results on blood alone, the reading for the sample
minus the blank reading was read. from the standard curve and. the
resulting figure multiplied by 20 to obtain mcg of thiamine per 100 ml
of blood.
For example, if one
obtained. a uluorometer
reading of 10.5
(which is the reading for the sample minus the reading for the blank)
then the concentration of thiamine as
read from the curve was
0.39 meg
of thiamine in 5 ml of blood or 7.80 meg of thiamine in 100 ml of blood.
In a personal communication, Dr. Friedemann stated that be calculated
his values in the same manner as above.
3A1.
CEAPTIJR I'V
ThiEDEMA1N
RESULTS AND DISCUSSION OF TUE STUDIES IN WHICH
AND ICMIEO IAN }EIETHOD WAS USED FOB TEE DETERMINATION ,Q
BLOOD THIAMIUE
The daily values for total Calories, non-fat Calories and. thiamine
See the footnote in.
in the diet were calculated from food tables.
Table 28 in Appendix II.
Non-fat Calories were calculated by u1ti-
plying the number of grams of protein and. carbohydrate in. the food.
These daily values, in. addition. to the ratio of non-fat Calories
by i.
to total Calories, thiamine to 1000
fat Calories are given in Table 28
Calories and. thiamine to 1000 non-
in Appendix II.
The amount of thiamine in the blood was determined every 5 days.
A summary of results for both
Study I and Study
Table A+ and. Figures 2 and 3.
The blood thiamine value for each period
II are given in
refers to the determination of blood. thiamine from a fasting blood
sample taken the morning following that period, for example, on February 9, 5 ml of blood were taken by venipimcture and. analyzed for
thiamine content in order to determine whether or not the previous
5-day (February L
through February 8) dietary
experience
had influenced.
the concentration of thiamine in the blood.
The blood
thiamine values
L.9l to 10.85 mog per cent.
for the subjects in. Study I ranged from
These subjects were all
girls.
From the
beginning to the end of the experimental period, there was a loss in
blood
thiamine for
all of the subjects in Study I.
There was also a
decrease in the mean intake of thiamine expressed in terms of mcg per
1000 Calories and meg per 1000 non-fat Calories as shown in. Table k and.
Table 4
THE CONCENTRATION OF THIAMINE IN THE BLOOD IN RELATION TO THE MEAN INTAKE OF TOTAL AND NOR-FAT CALORIES,
RATIO OP TOTAL TO NON..FAT CALORIES, THIAMINE INTAKE IN TERMS OF MCG PER DAY, MOG PER 1000 CALORIES,
MOG PER 1000 NONPAT CAL RIES FOR EACH GROUP OP 5 DAYS FOR AN EERIMENT.AL PERIOD OP 30 DAYS
Study I
Thiamine Inta
Ca1oio_Intake
1te
Subject
Total
NPC
Ratio of
NPC to
Total Cal
1947
lorma
arbara
61
6
Oct.
Oct.
Oct.
Nov.
Nov.
Nov.
18-21
22-26
27-31
1 - 5
6 -10
11-15
2195
2468
2201
2063
2472
2500
13324.
1324
1524
1559
64
62
Oct.
Oct.
Oct.
Nov.
Nov.
Nov.
18-21
22-26
27-31
1 - 5
6 -10
11-15
2163
2487
2302
2244
2728
2571
1356
1639
1509
63
66
65
114.34
64
1697
1599
62
62
1599
1410
6/+
63
Per 1000
Per Thay
Ca].
Per 1000
NYC
mog
Thiamine
In Blood
meg
og
meg
1205
1228
1191
1026
1161
1039
546
518
545
499
478
417
901
788
849
9.57
9.76
77/+
7.52
7.72
6.12
1236
1323
1282
1134
1239
1041
570
550
558
510
461
412
911
823
773
665
85/4.
79/i.
737
654
8.814.
9.40
9.93
7.12
6.92
6.12
4.91
Table 4
Study I (continued)
te
Subject
Caloric_Intake
Ratio of
NPC to
Total
Cal
Total
O
1947
larion
Roberta
Beesie
Oct.
Oct.
Oct.
Nov.
Nov.
Nov.
18-21
22-26
27-31
1 - 5
6 -10
11-15
1971
2244
2179
2056
2539
2339
1216
1493
1425
1344
1600
1481
62
67
66
6
63
Oct.
Oct.
Oct.
Nov.
Nov.
Nov.
18-21
22-26
27-31
1 - 5
6 -10
11-15
2110
2275
2192
2062
2542
2449
1280
1461
1437
1322
1589
1524
61
Oct.
Oct.
Oct.
Nov.
Noy.
Nov.
18-21
22-26
27-31
1 - 5
6 -10
11-15
2191
2374
2137
2099
2377
2220
1306
1508
1388
1329
1531
1396
60
64
65
64
64
65
611.
63
6
64
65
61+
Thiamine Intake
y
Per
mcg
1100
1148
1224
1015
1192
929
Per 1000
Per 1000
Gal
NJ'O
mog
meg
531
565
495
476
904
787
861
755
750
634
10.85
9.93
9.03
5.47
6.72
6.32
9.93
10.12
8.09
9.02
8.28
5.91
9 93
9.57
6.92
7.12
7.52
6.72
1+08
1125
1225
1252
1062
1260
971
534
1+04
887
540
876
801
799
640
1136
1176
1168
1031
1231
895
519
504
872
791
550
572
514
504
51+9
81+6
493
528
776
80?
647
411+
Thiaaino
In Blood
meg
0'
Table 4 (continued.)
Stu4 II
Oa]jrie_Int-ke
Ratio of
]te
Subiect
IWO to
Total
IWO
Total Cal
1948
Tom
Per 1000
Per 1000
Cal
IWO
meg
meg
meg
meg
2225
538
513
448
911
10.35
12.00
12.27
Per 1y
4103
2449
60
Feb. 14 - 8
14.54.5
4.912
59
59
2303
Feb. 9 -13
2698
2873
Feb. 14-18
Feb. 19-23
Feb. 24-28
4942
4632
4351
2858
2812
58
61
1449
776
14.51
7144
25144
59
2216
2082
1838
427
728
63
61
57
Feb. 114-18
14.999
2537
2788
2991
2889
Feb. 19-23
Feb. 24-28
5158
4572
3014.1
2578
58
59
56
2290
2280
2242
2218
2228
1738
553
Feb. 9 -13
4082
4636
5298
382
892
823
745
768
739
680
Jan. 31
Jan. 31-Feb. 3
3671
2215
61
2015
546
908
- 8
9 -13
3943
4363
2376
2504
60
57
1898
1865
797
752
Feb. 14-18
Feb. 19-23
Feb. 24-28
3909
2282
2280
2192
59
59
59
1678
1722
1429
481
431
4.30
Feb.
14.
-8
Feb. 4.
Feb.
Thiamine
In Blood
9.30
Jan. 31
Jan. 31-Feb. 3
Jan. 31
Jan. 31-Feb. 3
iesley
Thamine_Intairs__________
2194.
861
762
8.00
11.18
13.08
8.70
384.7
3709
496
4.23
444
1432
4448
388
737
767
657
8.0
8.314.
10.00
7.32
9.30
12.00
9.00
10.32
11.01
11.16
8.70
10.32
15.32
Table 4
Study II (continued)
ect
.te
Caloric_Intake
Ratio of
NYC to
NYC
Total Cal
Total
Jan. 31
Jan. 31-Feb. 3
- 8
Feb.
Feb. 9 -13
2638
3630
Cal
meg
meg
1474
1675
1949
2097
2105
1626
595
61
59
58
58
60
60
1597
63
63
62
63
979
863
696
736
723
678
15144
59
2129
2601
59
4141J4.
Feb. 114-18
14.187
214.75
Feb. 19-23
Feb. 24-28
4375
3932
2631
2280
2868
2261
3158
3204
3294
3087
1750
1606
1778
1695
1588
1454
1663
1012
1109
1041
1003
930
1106
14.
eith
Jan. 31
Jan. 31-Feb. 3
Feb. 14. * 8
Feb.
Feb.
Feb.
Feb.
Ruth
9 -13
14-18
19-23
24-28
Jan. 31
Jan. 31-Feb. 3
Feb.
14. - 8
Feb. 9 -13
Feb. 114-18
Feb. 19-23
Feb. 24-28
123
1830
1853
1991
1858
Per 1000
Per ]y
19148
on
Thiamine Intake
59
59
60
58
10214W
1227
1294
1280
1156
Thiamin
In Bloo
Per 1000
NFO
mog
mog%
1481
1025
783
755
852
800
14.16
720
10.70
10.70
10.85
9.30
9.15
11.01
14.20
56
450
388
405
388
923
767
670
699
649
626
9.30
11.01
9.00
9.68
8.00
11.32
13.93
14.63
1I45
502
378
8.70
614.
67
4114.
961
802
671
471
525
471
764
825
618
599
504
9.O
10.00
11.00
8.35
13.93
L
U,
FIGURE 2. IIEAN 5-DAY TH/Af1/NE INTAKE AND
BLOOD TH/At1INE VALUE5 FOR 5 SUB JECT5 IN STUDY
t
4-
-C'-)
OCT
1MEAN
NOV
DITES OF EXPERIPIENT
-OAY TN/A1INE IWTAKE FOR PERIOD I
tA)
"0
F/CL/RE 3. IIEAN 5-DAY 77-/IA/I/NE INTAKE AND
BLOOD TH/A11/NE VALUES FO/ 6 .SLJ5JEC TS IN STUDY 1T
-.-
9
JAN FEB
DATES OF EXPERItIENT
4(IEAM u-DAY THIAflINE INTAKE FOR PERIODI
Figures 2 and 3.
The average values for thiamine intake for Norma and Marion for
the last three 5day periods, November 1 through 5, November 6 through
10 and November 11 through 15, were below the National Research Council
recommended allowance (1948) of 500 meg per 1000 Calories.
Barbara's
intake was below the recommended. allowance for thiamine during periods
November 6 through 10 and
November 1]. through 15.
The same was true for
Bessie during the periods November 1 through 5 and November 11 through
15 and. for Roberta in the last period only, November 11 through 15.
There appears to be a relationship
between the loss of blood.
thiamine and. the decrease in thiamine intake in terms of meg per 1000
Calories in 4
of the
5 subjects as can be seen in Table 5.
table 5
THE 10RESE OF MEAN VAItIES FOR THIAMINE IN TEE BLOOD,
DECREASE OF THIAMINE PER 1000 CALORIES AND DECREASE IN
THIAMINE PER 1000 NONFAT CALORIES DURING STTJDY I
Bessie
3.21
105
225
Norma
3.45
129
236
Roberta
4.02
130
247
Barbara
4.49
158
257
Marion
4.53
149
270
42
The relationship is even more marked. between the loss of blood thiamine
and the decrease in thiamine intake in terms of mcg per 1000 non-fat
Calories.
That is, the greater the decrease in thiamine intake in
terms of mcg per 1000 non-fat Calories the greater is the loss of
blood thiamine.
Williams and. Spies (1938) have suggested that the
ratio of thiamine to non-fat Calories is a more accurate index of the
adequacy of the intake of thiamine than the ratio of thiamine to total
Calories.
The ratio of thiamine to non-fat Calories appears to be the
one most closely
related to
the values for thiamine in the blood. for
the subjects in Study I.
The Food and. Thitrit ion Board of the National Research Council
(1948) recommends that for
adolescents 30
to
per cent of the total
Calories should be provided in. the form of fat and for adults 20 to 25
per cant of the total Calories should be provided in the form of fat.
This was met or exceeded
in all cases
as may be seen in Table 2
in
Appendix II.
As may be seen in
in Study I
Figure 2, blood
stayed in. similar
thiàmine
values
for each subject
relative positions, e.g., Marion had the
highest blood thiamine value at the beginning of the study and. next to
the highest at the end of the study even though she lost the most in
blood thiamine during the 30-day period.
Bessie was next to
highest at the beginning of the study and the highest
study.
at
the
the end of the
Barbara had the lowest blood. thiamine value at both the
beginning and end of the study.
The boys in Study II bad higher values for thiamine in the blood.
than did the girls in Study I.
The values for thiamine in the blood
for the boys in Study II ranged from 8.00 to 15.32 meg per cent.
The
one girl in Study II had blood thiamine values ranging from 8.35 to
13.93 meg per cent.
The results for the
subjects in Study II do
not indicate the 88
relationship, as do those in Study I, between thiamine intake per 1000
Calories or thiamine intake per 1000 non-fat Calories and thiamine in
the blood as can be seen in Table 6.
There was an increase in the
concentration of the thiamine in the blood even though there was a
decrease in the
mean thiamine
intake from the beginning to the
end of
the experiment.
Table
6
THE INCREASE OF MEAN VALUES FOR THIAMIHE IN THE BLOOD, AND
DECREASE OF TRIAMIHE IETAXE PER 1000 CALORIES AIW DECREASE OF
THIAMIHE INTAXEPER1000 NON-FAT CALORIES DURING STUDY II
3.30
171
212
Don
3.50
179
305
Tom
3.78
11].
183
Keith
Jf.63
297
178
Rith
5.23
128
343
Wesley
6.32
158
251
ve
When the average values are considered, Tomts intake was wellabove
the recommendEd
al1oance of
thiamine per 1000 Calories in all periods.
The intakes of Dave, Wesley and Don wore above the recommended
allowance for thiamine in. all periods except that from
28.
through February
bruary 2k
KeIth's thiamine intake was somewhat
lower than
the recommended allowance in all periods except the first one.
general, all the boys except Keith
Ingested
In
much more liberal amounts
of thiamine with respect to total Calories than did the girls in. Study I.
Since the recommended allowance for thiamine provides a 100 per cent
margin of safety and the subjects
in Study II
in many eases ingested
higher amounts than the recommended aflowanoe, it woild appear that
they obtained sufficient thiamine throughout the 30-day period.
Ruth lost 11 3/Li. pounds on the 30-day study as a result of
voluntarily ingesting an average of less than 1800 Calories per day
throughout the study.
Her mean intakes of thiamine from food were only
slightly below 500 meg per 1000 Calories, the recommended allowance.
In
addition, some of her energy reqiirement must have been met from body
stores of fat.
Since Ruth was not maintaining weight the amount of
body fat metabolized would need to be considered along with
eaten.
the food.
This would. change appreciably the ratio of thiamine to Calories
and. would tend. to result in a "sparing action on thiamine and may be a
partial explanation for the increase of thiamine in. the blood despite
a lowered thiamine intake.
4.5
CHLPTER V
PROCEDURE ICR TEE EXPERIMENTAL STUDY IN WHICH IT WAS PLAN1ED TO USE
TEE BURCE METHOD FOR THE DETERMINATION 01 BLOOD THIAMXRE
PLAN OF TEE STUDY
There are few data available concerning the concentration of
thiamine in the blood and. no data have been obtained which indicate
whether or not there are variations from day to day in the concentration of thiamine in the blood when subjects are maintained, on a
constant intake of thiamine.
The Friedernann and Kmieciak method.
requires 5 ml of blood obtained by venipuncture and the method takes
2 days to complete the analyses.
This of necessity limits the frequency
with which determinations can be made both from the standpoint of time
to carry out the analyses and the objection to frequent venipunctures.
Because of the above reasons it seemed of interest to carry out
analyses using an unpublished micro-method. that has been developed by
Dr. Helen Burch (1948).
This method has the advantages of using only
50 cmm of blood obtaifled from a finger puncture and the fact that the
analysis can be completed in one day.
A metabolism study1 using human subjects was planned in order to
determine the daily values for thiamine in the blood when subjects were
maintained. on a controlled diet over a period of 52 days.
The study in which the Burch method was attempted. involved. analyses
1
For the support of this research, appreciation is expressed to the
Research Corporation of New York City for a grant from the
Williams-Waterman Fund for the Combat of Dietary Diseases.
of both blood and urine so that the responsibility of the subjects
included (1)
(2) the
eating the diet prepared for them in the laboratory,
collection of 214-hour urine
samples, (3)
providing fasting
blood samples by finger puncture each morning and (Lf) taking
ments of crystalline thiamine and ascorbic acid.
supple-
A copy of the
directions given to the subjects is given in Appendix III.
In general, the subjects arrived at the nutrition research
laboratory at 5:45 A.M. each day.
The subjects weighed themselves
(without shoes) and. recorded their weights in the research book
provided for that purpose.
The individual voidings and total volume
of urine excreted. in the preceding 214-hours were recorded iu addition
to menstrual period, number of bowel movements, unusual exercise or any
observation that might aid in later interpretation of the data.
The 24-hour
period for the collection of urine began aster the
first voiding on the first day and included the first voiding on the
next day.
Twenty-four hour urine specimens were collected each day
throughout the experimental studr.
Each voiding was divided so that
one-half of the total volume could be kept without a preservative and.
to the other half of the volume a preservative (io% by volume of
metaphosphoric acid, in 2
sulfuric acid.) could. be added.
All of the
urine samples were kept in the refrigerator or in the cold. and were
analyzed on the day following the collection.
The preserved. urine was
analyzed for the excretion of thiamine and ascorbic acid., and the
unpreserved urine was used. for the croatinine and bomogentisic acid
analyses.
Fasting blood samples were taken by finger puncture for the
L17
analyses.
The hand. was warmed under running warm water, dried and the
finger punctured deeply enough by means of
a
Bard-Parker blade to
obtain 3.5 ml of blood to provide a sufficient volume of blood. for the
analyses.
These analyses included the determination of thiamine in the
blood, hematoorit, total and reduced ascorbic acid in the serum and.
plasma by two different methods and the determination of the ascorbic
acid. content of the white-cell-platelet layer.
For all of the above
determinations it was essential that the punctured finger was not
squeezed except, if necessary, for the
collection of
serum.
Supplements of crystalline ascorbic acid and. thiamine hydro-
chloride, dissolved in water, were taken after all of the blood samples
were
obtained.
laboratory since
Breakfast was served at tables in the nutrition research
all of the subjects were busy
in the laboratory in the morning.
carrying out analyses
Lunch and. dinner were eaten in one
of the dining rooms in the Home Economics building.
Even though the same foods were eaten each day over a period of
7 weeks
the
foods were eaten readily by all of the subjects.
They
maintained good general health throughout the study and no subjectl.ve
symptoms of deficiency were noted.
EXPERIh4ENTAL DIET
The experimental diet consisted. of a basal diet with additions to
it planned in units.
The plan was to set up a basal diet providing
approximately 1000 Oalories and 300 mcg of thiamine and. a unit to
provide approximately 500 Calories and 150 meg of thiamine.
The
determined values for the basal diet (Table 7) provided 1021 Calories
and 305
meg of thiamine and. each
and. 153 meg of thiamine.
unit (Table 7) provided 513 Calories
Yor the sake of clarity these diets will be
referred to using the approximate values.
The values for protein, fat, carbohydrate and. total Oalories were
Nonfat Calories (NYC) wire calculated by
obtained. from food tables.
multiplying the total number of grams of protein and carbohydrate by 1+.
The foods were bought in case lots at the beginning of the study
in amounts sufficient to
last
The biscuits and. cookies
(Table 8)
through the entire experimental period..
were prepared. using unenriched. flour.
Samples of the foods were analyzed for their content of thiamine and
ascorbic acid.
The method of Hennessy and Cerecedo (1939) as modified.
by Merck and Company (191+1) was used for the analysis of thiamine and.
the method of Loeffler and Pont ing (191+2) was used for the analysis
of the ascorbic acid content of the food..
Studies on ascorbic acid
the thiamine study.
metabolism were carried. out along with
It was planned that the subjects would be
main
tained. on a constant intake of 25 mg of ascorbic acid. per day through
out the experimental period..
ascorbic acid.
The basal diet provided 9.16 mg of
A daily supplement of 15.81+ mg of crystalline ascorbic
acid was given to provide
a
total of 25 mg of ascorbic acid per day.
The subjects were told. that they would be allowed 3 days to decide
on the level of Calories that they would choose to eat.
Three of the
subjects started at a level of 2500 Calories, i.e., the basal diet
plus 3 units, and 2 subjects started at a level of 2000 Calories, i.e.,
49
Table 7
COQOSITI0N OF BASAL DIET
C00SITI0N OF tflIT ADDED TO BASAL DIET
gin
gui
gui
gin
Ascorbi
Total Non-fat
Ascorbi
acid
mg
Total Non-fat
tProtFatCarb0alQTAineS
mcg
gui
gin
gin
meg
gin
mg
Milk, evap.1
100
7.0
7.9
9.9
139
68
21
0.03
Biscuits4
55
3.0
7.8 21.0
166
96
12
0
Oarrots, canned3
100
1.0
0.3
7.6
37
34
14
3.1.12
Cookie4
48
2.11
9.3
215
131
11
0
&ef, round'
100 19.3 13.0
0
194
77
36
1.26
Bu.tter1
10
0.1
8.1
0
73
0
0
0
Pears, canned'
100
0.2
0.1.
18.4
7
74
7
0.16
Sugar, white1
0
0
5.0
20
20
0
0
Pruxies, dr., stewed2 100
1.7
0.5 53.3
224
220
51
3.23
Wheat
2.5
1.0
5.0
39
30
130
0
100
1.0
0.1
3.3
18
17
46
1.06
8.0 26.2 61.4
513
277
153
0
108
105
11
0
85
29
32
0
Gr. beans, canned3
Cream of wheat1
30
3.5
0.3 22.8
Eggs, E.P.3
54
6.9
6.2
0.L
Cheese, cheddar1
30
7.2
9.7
0.5
118
31
9
0
6
1.5
0.6
3.0
23
18
78
0
49.3 38.7119.2 1021
673
305
Wheat germ'
1
2
erm1
5
10
9.16
Values from U.S.D.A. Misc. Pub. 572, except for thiamine and ascorbic acid.
Calculated, on the basis that 75 gin of dried prunes is equivalent to 100 gin cooked prunes (values from U.S.D.A. Misc. Pub. 572, except
for thiamine and ascorbic acid.).
3
4
5
6
Values from U.S.D.A. Circ. 549, except for thiamine and ascorbic acid.
Values calculated from ingredients (values for ingredients obtained from U.S.D.A. Misc. Pub. 572, except for thiamine and ascorbic acid).
Foods were analyzed for thiamine content by Miss ilsi Usuan Yu.
Foods were analyzed for ascorbic acid content by Miss Bessie Ivey and Miss Mel-lirig Wu.
Table 8
COMPOSITION OF BISCUIT MIX
Total Nonf atl
Fbod.
Flour unenriched.2
Crisco2
Salt(1T)
Baking powder, Royal
Water
Amount
Protein
Tht
gin
gin
gin
Cal
243
1620
0
0
0
0
0
0
0
0
71.3
185.9
500.9
3963
71.3
55
0
0
0
Cal
gin
500.9
660
180
15
400
1310
Carbohydrate
5.9
180.0
0
0
0
2289
Baked at 4500 for 12 minutes
COMPOSITION OF COOKIE MIX
Total Nonfat1
Food.
Flour uueririched.2
Sugar, brown2
Crisco2
Eggs (2)2
Salt(1T)
Soda(t)
Vanilla (iT)
Ainotuit
Protein
Fat
gin
gin
gin
gin
4.1
341.6
429.8
450
450
220
100
48.6
0
0
0
1220
Baked at 375
1
0
0
Carbohydrate
0
220.0
11.5
0
0
0
0
0
0
0
0
0
0
61.4
235.6
772.1
12.8
0.7
Nonfat Calories were calculated from
Values were
Cal
1598
1719
1980
158
0
0
0
5455
3334
for 10 minutes
the
total grams of protein and.
carbohydrate malt iplied. by 4.
2
Cal
obtained from U.S.D.A. Misc Pub. 572.
51
the basal diet plus 2 units.
After the second. day, however, all of the
subjects decided to eat 2000
alories1. Thus, for the rest of the
experimental study (1f9 days) all 5 subjects were
containing approximately 2000 Calories.
in grams, of the 5
subjects from
maintained on a
diet
The dietary intake expressed.
January 31, 19LI9, through March 21,
l9J9, is given in Table 9.
During the first 31 days (Period. I) the subjects were given a
supplement of kOO meg of thiamine
thiamine up to the Eational
hydrochloride to
bring the intake of
esearch Council's recommended allowance
of 500 meg of thiamine per 1000 Calories.
Daring the next 21 days
(Period. II) the subjects were given no additional supplement of thiamine
hydrochloride so their intake was reduced to approximately 300 meg of
thiamine per 1000 Calories (Table 10).
DESCRIPTI0T OP THE SUBJECTS
Five adult women were subjects during the 52-day study.
all moderately active and presumably normal2,
healthy
They were
individuals.
each
of the subjects also had. responsibility for some phase of the laboratory
work so all
had a more than usual interest in and understanding of the
details of the study0
height, weight at
A description
the beginning
of the subjects in
terms of age,
and the end. of the study, weight range
and average weight is given in. Table 11.
1
2
In
BWC's diet was slightly modified as can be noted. in. Table 10.
addition, this subject s diet was supplemented with a commercial
vitamin-mineral preparation (Stuart Formula) which contained at
least 2 mg of thiamine chloride.
BLD had had poliomyelitis when young, WO has nephritis.
52
Table
T
9
DIET CONSUMED BY 5 SUBJECTS ThOM
J.AEUABY 31, 19Lf9 THROUGH MARCH 21, 1949
BLD
BWO1
MLW
KHY
CAS
Cream of Wheat
30
30
30
30
30
Eggs2, B.?.
54
54.
54.
54.
Cheese
30
30
30
30
30
Green Beans
100
100
100
100
100
Prunes
100
100
100
100
100
Biscuits
55
n°'°
Cookies
48
48
48
.48
48
Meat
100
none
100
100
100
Carrots
100
100
100
100
100
Pears
100
200
100
100
100
Biscuits
55
55
55
55
.55
Cookies
48
48
48
48
48
100
100
100
100
100
Bu.tter
20
40
20
20
20
Sugar
10
15
10
10
10
Food.
Breakfast
Lunch
!ood.s consumed. by the subjects during the day
Milk
1
2
26
26
26
26
Wheat term
26
BWC had. some dietary restrictions and. took a commercial vitaminmineral
supplement. Although her intake was not the same as the others it
was constant throughout the stu&y.
each subject.
One egg was served. to
B.?., was 54. grams.
The average weight of the eggs,
.53
Table 10
COMPARISON OF THLMflTh II'ITAXE I4ITH CALORIC INTAKE FOR 5 SUBJECTS
ThIRING THE PERIOD JANUARY 31, 1949, THROUGH MARCH 21, 1949
Cioric Intake
Thiamine Intake
Ratio of
NFC1to
Prot
Fat
Oarb
3LD
65.3
91.1
WC
43.4'
Su1c,t
HEY
I
2
3
1+
5
4On-!at
PeriotI
McgOQ
Total
NEC1
242.0
2047
1227
59.94
86.6
244.4
1928
1148
59.54
65.3
91.1
242.0
2047
1227
59.94
1011
65.3
91.1
21+2.0
2047
1227
59.94
65.3
91.1
21+2.0
2047
1227
59.94
ca!OrleS
al
Period II
Ji000 ii
Mc
Mc ,Ji000 Cal
McOOO NEC
498
824
611
25874'
25705
494'
824
611
298
498
1011
l94
824
611
298
498
1011
494
824'
611
298
498
1011
494
298
22395
Thiamine content of the food. plus supplement of 400 mcg thiamine hydrochloride.
Thiamine content of the food only.
Thiamine content of the food plus a supplement of 400 mc of thiamine hydrochloride plus 2000 cg thiamine chloride from a commerca1
multiple vitaminmineral preparation (Stuart Formula).
Thiamine content of the food plus 2000 mcg thiamine chloride from a commercial multiple vitamin and miera1 preparation (Stuart Formul&).
5k
Table 11
DESCRIPTION OF TEJ SUBJECTS
Body Weight
Beg.
End.
of
Study
Weight
Range
lb
Average
Wei
lb
Age
yr
Heigjit
in
of
Study
lb
ELD
28
62 3/k
107 1/2
108 1/2
106 3/11-110 3/k
108 1/2
EWO
3].
62
116
112
111 3/11-116 1/k
113 1/2
314.
65 1/2
125 i/k
123
122
-126
123 3/k
36
6k
128
125
125
-128
126 1/2
14.2
68 1/2
114.9
114.7
1k6
-11.9
1k7
Subject
jyl
US
1
These subjects are Chinese.
lb
CH.PTER VI
EQUIPMET USED
The method of Burch (unpublished) was u.sed in studying a micro
method for the determination of thiamine in blood.
The equipment used.
for this proced.ire was as follows:
1.
BardParker blade, size 11
2.
Small vials having a capacity of approximately 1 ml.
These
were made from 10 mm glass tubing.
der number 2.
3.
Small corks to fit the vials dsoribed
Li.
Test tubes, 6 x 50 mm
5.
Pyrex test tubes, 10 x 75 mm, unselected
6.
Pyrex test tubes, 10 x 75 mm, optically matched tubes for use
in the microphotofimorometer
7.
I4icropipettea - 5, 20, 23, 50, 200 and 225 emm capacity.
These pipettes were made in the laboratory according to the
directions given by Bessey, Lowry and Brook (l9Li6).
'or
calibration of the micropipettes see Appendix IT.
8.
Buzzer - A buzzer was improvised. by using the rapi&].y
whirling nut on the wheel of an Amend grinder.
By touching
the bottom of the test tube against the whirling nut the
contents of the tube were violently agitated.
9.
10.
bber caps to fit the 6 x 50 mm test tubes
Metal racks to hold the 6 x 50 mm test tubes
11.
Wooden racks to hold the 10 x 75 mm test tubes
12.
Wire basket woven with string in
test
tubes upright and
order to hold
to separate
them from
the 10 x 75 mm
each
other while
in the incubator.
13.
Clinical centrifuge
1)4..
Parafi].m
15.
Syringe pipettos]. - to hold. 1 ml and 2 in]. hypodermic syringes
16.
Hypodermic syringes - 1 in]. and 2 ml capacity
17.
Hypodermic needles - 20
18.
Transfer
gauge
pipettes - made in the laboratory by making a
constriction pipette that wou.ld transfer about 1 ml of
solution.
19.
Incubator
20.
Jrrand micro-photofluorometer - An extra gelatin filter,
Jo. 2A (Eastman Kodak Co.) was
used to
reduce the fluorescence
resulting from the filters themselves (Lowry, 19Lf8).
21.
H-5 mercury vapor lamp
22.
Socket and. transformer for the H..5 mercury vapor lamp
23.
Irradiation rack - made in such a way that the 10 x 7.5 mm
tubes were irradiated while standing upright ectly 3 inches
from the filament of the H-5 mercury vapor lamp.
This set-up
is similar to that described by Bessey, Lowry, Brock and
Lopez (19k6).
1
Syringe pipettes were obtained from
Mr. H. Iff
Northern Tool and Instrinent Co.
l61_21 Northern Blvd.
flushing, New York
57
2L1.
Stop-.watch
25.
Automatic timer
26.
Volumetric pipettes
RAGTS USED
The reagents1 used. for the determinatIon of thiamine in the blood
by the Burch method were as follows:
1.
5% triohioroacetic acid.
2.
k
3.
Phosphatase - Either one of the following was used
potassium acetate
a.
Olarase was prepared by Dr. H. Bu.rch
b.
Acid. phosphatase was prepared from hux:n seminal fluid
by diluting one part of seminal fluid with 5 parts of
redistilled. water.
acetic acid..
The pH was adjusted to 5.0 with
This solution was centrifuged at high speed.
for one hour and. stored in the refrigerator.
Care was
taken to see that the preparation was kept cold but did.
not freeze.
k.
30% sodium hydroxide (carbondioxide free) * A saturated
solution of sodium hydroxide was prepared by adding 1100 grams
of C.P. sodium hydroxide to 1000 ml of water redistilled in
glass.
This solution was considered to contain 75 grams of
s dium hydroxide per 100 ml of solution (Hawk, Oser and
S mmerson, p. 123k, 19k7).
1
Dilutions were made to 30% sodium
All reaents were made up with water red.istilled. in glass.
hydroxide from this reagent.
5.
2% potassium ferricyanid.e - made up once each month
6.
5.5 N sodium dihyd.rogen phosphate containing 0.015% hydrogen
peroxide.
We used. 189.82 gin NaH2?0L1..
H20 plus 0.125 gin
hydrogen peroxide (sp.g. 1.1) made up to a volume of 250 ml
with water redistilled in glass.
7.
N-butyl alcohol - All n-butyl alcohol was redistilled. in an
all glass still collecting the 116 to 118
degree Centigrade
boiling fraction.
8.
Approximately 1.0! sulfuric acid from which approximately
0.1 N sulfuric acid was prepared
9.
Approximately 1.0 ! hydrochloric acid, from which approximately
0.1
10.
hydrochloric acid 'was prepared.
quinine sulfate stock standard - 100 mg quinine sulfate,
U.S.?., was made up to a volume of 1 liter with approximately
0.1 N sulfuric acid.
This solution was stored in the
refrigerator in an amber-colored 'bottle.
11.
quinine sulfate working standard - 1 ml of uiniue sulfate
stock standard was diluted to a volume of 200 ml with approximately 0.1 R sulfuric acid.
This reagent was prepared daily
and protected from the light until used..
12.
Thiamine stock standard - Exactly 50 mg of anhyd.rous
thiamine
hydrochloride (dried several weeks over sulfuric acid) were
made up to a volume of 500 ml with approximately 0.01
hydrochloric acid.
hydrochloride.
Thus, 1 ml contained 100 mcg of thiamine
13.
Thiamine working standard - 1 ml of thiamine stock standard
was diluted to a volume of 100 ml with approximately 0.01
hydrochloric acid.
Thus, 1 ml contained 1 mcg of thiamine
hydrochloride.
PERMINAT ION OP THIAMflIE IN THE BLOOD
The determination of thiamine in the blood was as follows:
Collection of Blood Sample
1.
A finger was lanced with a Bard-Parker blade.
The first drop
of blood was wiped off and the next 6 or 7 drops of blood
were collected in a small vial.
This amol2nt of blood would
give 3 to k samples if the collection and measuring of blood
was carried out without delay.
2.
The vial was stoppered. with a cork and inverted several times
so that the blood was well mixed.
Precipitation
3.
the Protein
Immediately after mixing, 50 cmm samples of the blood were
delivered into a 6 x 50 mm tube containing 225 cmm of 5 per
cent trichioroacetic acid.
11.
The tubes containing the blood plus trichioroacetic acid
wore buzzed immediately.
5.
After buzzing, the tubes containing the blood.-tricbloroacetic
acid mixture were covered with small rubber caps and allowed
to stand at room temperature 30 minutes after the last blood
was measured.
6.
The above tubes were buzzed again and. then centrifuged at
full speed. for 10 minutes to colete1y separate the precipitated. protein from the supernatant.
7.
200 emm of the supernatant wore drawn off into 10 x 7.5 mm
tubes.
Diestipn with Pbosbatase
potassium acetate were added..
8.
20 cmm of L
9.
23 cmm of Buroli clarase or 10 omm of acid. phosphatase were
added..
10.
The tubes were covered. with Parafilm, placed in tie wire
basket and incubated 2* hours in a humidity-controlled
incubator maintained at 37 to 38 degrees Centigrade.
11.
Blanks were prepared by adding 200 cmm of a diluted .5 per
cent trichloroacetic acid. solution (2.3 ml of 5 per cent
trichioroacetic acid plus 0.5 in]. of red.lstilled water) to
the 10 x 75 mm tubes.
20 cmm of potassium acetate and 23 cmm
of Burch clarase or 10 cinm of acid phosphatase were added..
These were covered with Parafilm and. incubated 2* hours along
with the blood samples.
12.
Standards were prepared by adding 5 cmiii of the thiamine
working standard solution to 200 omm of a diluted 5 per cent
trichioroacetie acid (2.3 ml of 5 per cent trichioroacetic
acid plus 0.5 nil, of redistilled water).
20 cmm of potassium
acetate and 23 cmm of Burch clarase or 10 emni of acid.
phosphatase were added.
These tubes were covered with
Parafilm and incubated 2* hours along with the blood samples.
ri
Oxidation to Thiochrome
13.
]3y
The entire amount in each tube was used. for oxidation.
means of a syringe pipette, 0.1 ml of a mixture of 30 per
cent sodium hydroxide containing 0.12 per cent potassium
ferricyanide was added to blood. samples, standards and. b1axks.
Tor the oxidation mixture we used. 1
potassium
ml of 2 per cent
ferricyanide and made it up to a volume of 25 ml
with 30 per
cent
sodium hydroxide.
This mixture was prepared.
immediately before use and was not kept more than an hour.
1)4.
with
The tubes were mixed at once by tapping
the finger for
exactly 15 seconds.
15.
Immediately after the 15
dihydrogen
seconds, 0.1 ml of
5.5
sodium
phosphate containing 0.015 per cent hydrogen
peroxide was added. with a syringe pipette.
a
syringe pipette.
16.
1 ml. of n-butyl alcohol was added with
17.
The tubes were buzzed for 15 seconds and centrifu.ged for
2 minutes to separate the layers.
The clear n-butyl alcohol
layer was transferred to an optically
matched 10 x
75
mrs tube
by means of a transfer pipette. A clean dry transfer pipette
was used to transfer each sample.
18.
All tubes were read in the Jarrand. micro-photofluorometer
which was set at 90 with the quinine sulfate working standard..
Diaphragm opening number 6 was used on the Farrand. microphotofluorometer.
19.
After reading the optically matched tubes in the microphotofluorometer, all the tubes were irradiated for 30 minutes
62
3 Inches from the filament of the R-5 mercury vapor lamp.
The H-5 lamp was allowed to warm up for 10 minutes before
the tubes were irradiated.
20.
Tho optically matched tubes were then reread in. the micro-
photofluorometer.
Calculations
21.
The irradiated reading was subtracted from the reading before
Irradiation and then the average blank reading subtracted
from the remainder to give the final reading which was used
in. the calculations, as shown in Table 12.
Table 12
TH0D OF DETERMINING TEE AVERAGE CORRECTED BEADING
USED IN TEE üALOULAT ION OF BlOOD TEIAMflE
Tube Contents
Read.
Before
Read.
After
Read.
Average
Before- Blank
Irrad..
Irrad..
After
(Col.1i-Col.5)
Average
Corrected
Reading
8.95
Corrected
Reading
Irrad.
Blood (BC)
L4.2.25
30.00
12.25
1.75
10.50
Lf2.50
32.00
10.50
1.75
8.75
Li.6.00
35.00
11.00
1.75
9.25
Li.2.25
32.00
10.25
1.75
8.50
112.00
32.50
9.50
1.75
7.75
63
supernøt
b1ood(°
\ total yalume
ml biood per aliquot
rnl
ml blood per aliquot
(0.05) (2Q
275J
0.O36 ml
(100 ' = meg % thiamine
( mince thiamine \ f Corrected read. sampi
'4m]. blood/aliquOt I
Corrected read. stan.dardi '4.1000)
= 2.05 meg
thiamine
CEAPFER VII
D.ETRMflt&TION 91 EEMTOORfl'
ItEED
R THE
TERMII(LTIOI OF HEMATOORIT
Gorham, Abels and Robbins (19k2) and Florijn and Smite (1948)
foud. most of the thiamine in the blood was contained in the blood
Hennessy (19k?) states that "The thiamin of blood is
cells.
virtually all in the cellular elements and. a mere statement of the
thiamin content of whole blood is therefore almost meaningless....
An approximation
can be made in the absence of leukemia if at least
the hematocrit value is given in addition to the thiamin content of
the whole blood."
For
this reason it was planned to determine the
beinatocrit in addition to total thiamine
in order
to aid in the later
interpretation of the data.
The equipment used for the determination of the hematoorit
included the following:
1.
Small rubber vaccine bulbs
2.
Spot plate
3.
Capillary tubes
14
Pyseal cement
5.
Alcohol lamp
6.
Clinical centrifuge
7.
Micropipette, approximately 1 c
capacity
65
F1
PROCEDURE FOR DETERMINATION OF HEMLTOCRIT
The procedure for the determination of the hematocrit was as
follows:
Selection of Pubes
Oapillary tubes were selected that were of uniform bore (3mm)
and 4 inches in length.
The ends of the tube were brushed with a
piece of screen if necessary so that they were smooth.
These capil-
lary tubes were washed and dried so that they were chemically clean.
Using a micro-pipette, 1 cmm of heparin solution (Roche-Ornon,
10 mg in 1 ml) was placed in the capillary tubes one-half inch from
the end and. the tubes were allowed to dry.
Collection of Elood.
Rubber bulbs were placed on the ends of the capillary tubes
opposite the beparinized. ends.
After the finger had. been lanced with
a Eard-Parker blade and. the sample collected for the blood thiamine
determination, the blood (about 0.1 ml) was allowed to flow up the
capillary tube until it reached about one-fourth inch from the end..
The blood was mixed with the heparin by using the small rubber bulb
to express the blood in and out of the tube into a spot plate.
mixing was carefully done so that no bemolysis would occur.
This
The blood
was finally drawn into the tube and. the unheparinized end sealed with
Pyseal.
CentrifuEation
The tubes were labelled and. then centrifuged one hour at full
speed in a clinical centrifuge in the cold.
Our centrifuge was kept
in a refrigerator throughout the experimental period. After the one
hour centrifugation,
the tubes
were removed and
length of the packed cells and the total
read. by reading the
length of cells plus plasma.
Cal culat ion
The length of the red blood.
length of the
cell layer divided by the total
cells plus plasma and. multiplied, by 100 gives
the
per
cent of the blood volue occupied by the red. cells.
RESULTS OP THE DTERMINAT ION OP HEMATOOHIT
The values for hematocrit were to be
of the results of the
the Barch method.
used in the
interpretat1on
concentration of thiamine in the blood using
Since the latter values have not been
all of the data for hematocrit are listed in Table 29
determined,
in the appendix.
67
CHAPTER VIII
In spite of the fact that considerable preliminary work was
performed before the study started our blood thiamine
a1ues were
always significantly lower than results (I+.8 to 6.5 meg per cent)
obtained by Dr. Bureh in her laboratory, than other values reported
in the literature or our
method.
results
with the Friedemann. and Kmieciak
It was planned to follow the concentration of thiamine in the
blood during the first few days of the study when the subjects were
receiving a
own amount of thiamine to observe whether any change
occurred in the blood thiamine value, but they continued at the same
After 10 days of the experimental period it was decided to
low level.
freeze the blood supernatant (the 200 0mm of supernatant removed after
precipitation of the protein and centrifugation) until the problem
was solved.
Fasting blood samples were obtained and deproteinized.
each day throughout the study.
Aliquots (200 cmm) of the supernatant
were frozen in dry ice and are still being held
frozen
until the
problem is solved before the thiamine analyses are attempted.
METHODS USED FOR FURTHER STUDY OP THE BUBCH METHOD
Varying the .&mounts of Potassium Acetate Used
The determination of the pH of the blood supernatant using
varying
amounts of potassium acetate - One of the first questions that came to
ri
mind, was whether or not the pH was optimum for the enzyme hydrolysis.
On Janunry 18, 1949, Dr. Buroh had suggested that a check be made on
the pH after the neutralization with potassium acetate to be sure that
the pH was between 4.6 and. 4.8.
Sinoe we were still having difficulty,
on March11, 1949, D. Bu.rch suggested that we try less potassium
acetate resulting in a pH of about 4.4 for enzymatic hydrolysis in
order to decrease the salt concentration.
This suggestion was made
since it is known that high sodium acetate inhibits the enzyme.
inhibition might be overcome by dilution.
This
The various amounts of
potassium acetate added to the blood supernatant and. the resulting pH
are given in Table 1'.
The determination of the pH of the blood supernatant plus varying
amounts of potassium acetate and acid phosphatase - Since the ensyme
itself is adjusted to a pH of about 5.0 the effect of the ad.d.itin of
10 cmm or 23 cmm of acid phosphatase (P) on the pH after addition of
varying amounts of potassium acetate was observed.
The following
results (Table 14) indicate that little change in pH occurred with the
addition of the enzyme.
Determination of thiamine in fasting blood when varying amounts of
potassium acetate were used - In no case did the results indicate a
much higher blood thiamine value due to the possibility of a more
optimum pH by the addition of varying amounts of potassium acetate.
The results on March 18, 1949, when 15 omm of potassium acetate were
used, suggested that there was a change in the blood value but
another study on March 19 did. not confirm this (Table ii).
Table 13
THE pH OF THE BLOOD SUPEPEATANT WHEN VARYING AMOUNTS
OF POTASSIUM ACETATE WERE ADDED
Amount of
Potassiwn
Acetate
cmm
pH
I.te
(1949)
15
4.33
BWC
Mar. 18
17.5
4.53
BLD
Mar.
17
20
14.72
14.72
MLF
BLD
EWO
BWC
27
27
18
18
4.501
CAS
Jan.
Jan.
Feb.
Feb.
Mar.
23
4.92
NLF
Jan. 27
28
1+.821
GAS
GAS
GAS
Mar. 10
Mar. 10
Mar. 10
4.68
4.71
4.621
1
Subject
10
Those d.eterrnination.s of pH were made with email micro-pR-beakers
(Dietz, 19148). These micro-beakers reauired only a drop or two
of solution but were a little difficult to handle to get
consistent results.
It is suggested that for the instrument in
the nutrition laboratory the depressions into which the
solution is placed be hollowed out still further. This would
require a slightly larger volume but would probably give more
reliable readings.
70
Table 14
TEE pH OF TEE] BLOOD SUPEBNATANT AFTER ADDING VARYING AMOUNTS
OF POTASSIUM ACETATE AND ACID PHOSPHATASE
Amount of
Blood
Sernatant
Potassium
Amouxit of
pH
Acetate Added
.AP Added
cmm
cinm
cmx
200
200
200
200
15
20
23
28
4e53
200
200
200
200
15
20
23
28
Li.53
.83
Li..95
5.12
4.86
4.90
5.12
pH
10
10
10
10
k.51
k.75
23
23
23
23
4.50
4.74
4.85
5.05
95
5.02
Table 15
DETERMINATION OP BLOOD THIAMINE WREN VARYING AMOUNTS OP
POTASSIUM ACETATE WERE USED
Amount of
Potassium
Acetate
cmx
Blood
Thiamine
BWC
15
5.27
non-fasting blood
23 cmx clarase
extraction with
isobutyl alcohol
Mar. 18
EWO
15
5.29
Non-fasting blood
Mar. 18
Su.bject
Remarks
mog%
te
(1949)
10 cmx .AP
extraction with
isobutyl alcohol
71
Table 15 (continued)
Arnoimt of'
?otassi
Acetate
cmm
Blood
Thiamine
BLD
15
2.83
BWC
MLW
OAS
15
15
15
15
3.93
1.96
2.03
2.93
BLD
17.5
1.28
Subject
Remarks
mcg%
Date
(19J+9)
extraction with
isobutyl alcohol
I'
Mar. 19
It
0
I'
non-fasting blood
Mar. 15
23 cinm elarase
BLD
17.5
2.16
BLD
17.5
2.31
Mar. 17
non-fasting blood
Mar. 15
10 cmm P
BLD
17.5
1.96
Mar. 17
CAS
20
2.66
Mar. 10
BLD
20
1.19
Mar. 13
BWC
20
20
20
2.5A'
CAS
20
3.85
Mar. 22
QAS, HB
20
20
20
2.82
3.01
Mar. 2
20
20
20
20
2.61+
BLD
23
1.73
Mar. 13
BLD
28
2.48
Mar. 13
(pooled)
BWC
2.BLi.
use of new reagents
II
Mar. 29
ft
It
2.76
Ii
2.91+
2.69
2.80
3.19
Mar. 31
0
II
'I
II
72
The determinations on March 18 were carried, out on blood collected at
5 P.M. (non-fasting) and iso-'butyl alcohol was used. for the extraction
instead of n-butyl alcohol.
The use of iso-butyl alcohol did not
explain this difference in value, however, as will be discussed later
in this section.
Twenty cmm of potassium acetate was finally decided upon as the
volume to be used in future analyses because that was the amount
suggested by Dr. Btu'ch and. because more or less than that amount was
of no advantage.
The Use of Different Trichioroacetic Acid Reagents
The use of redistilled trichioroacetic acid. - On March 27, 1949, it was
learned. that redistillod trichioroacetic acid was used in the original
method since the commercial product varies widely.
Coleman and. Bell1s
trichioroacetic acid was used. for all of the experimental blood samples
Both
that are being kept frozen until the problem of method is solved.
Dr. H. Burch and Dr. 0. H. Lowry have given directions for the
redistillation of trichloroacetic acid. in an all glass still under
reduced pressure.
An attempt was made to redistill some triohioroacetic
acid. with the assistance of Dr. B. B. Christensen of the Chemistry
Department.
Some redistilled trichioroacetie acid. was obtained but the
resulting product was not as pure white as was the Coleman and Bell
reagent.
It was felt that further work should be done on a set-up for
the distillation of this reagent since the method is more involved.
than it would. appear to be.
Dr. 3. H. Haag of the Agricultural Chemistry Department gave us
some trichioroacetic acid that he had. redistilled in his laboratory
73
several years ago which was used. in the following experiment.
The
concentration of thiamine in the bloo& was determined, when the protein
of 50 cmm blood sales was precipitated by 5 per cent redistilled.
trichioroacetic acid. and when 50 cmm aliq,uots of the same blood. were
precipitated by 5 per cent trichioroacotic acid as we had prepared it
from the trichioroacetic acid. which had not been red.istilled..
difference in values was noted.
No real
In fact, in these two analyses a lower
value was obtained for blood thiamine with the redistilled. trichioroacetic acid..
Use of different brands of trichioroacetic acid. - Coleman and Bell's,
Baker's and Mallinckrod.t's trichioroacetic acid were used to determine
blood. thiamine and no difference in values was observed..
Teats for Enzyme Activity
Determination of blood. thiamine with and. without the use of olarase -
The values obtained in the nutrition laboratory were mu.ch lower than
Dr. Burch had obtained with the micro-method so an. answer to the
discrepancy was sought in the q,uestion of enzyme activity.
The clarase
used. with the micro-method was an especially pnrified preparation from
Dr. Bu,rch's laboratory.
Coerôial clarase, without purification,
produces too high a blank for the micro-method..
Before cocarboxylase (thiamine pyrophosphate) was obtained, the
activity of the Burch clarase was tested. by noting a difference in the
reading for blood. thiamine when clarase was added to some blood
s'a.pornatauts and not added to others.
1
This procedure was followed in
Throughout the discussion fasting blood. was used. trn].ess otherwise
stated..
74
5 different experiments at varying times during the year and. in every
case the corrected. reading was zero or almost zero when no clarase was
added to the blood. supernatants.
When clarase was added to the blood.
supernatants the blood thiamine determinations were similar to those
already reported in this thesis.
Varying the amount of clarase used. The Bu.rch clarase preparation had.
been prepared over a year before this study began.
It was thought
possible that the activity of the enzyme had. decreased or that 23 cmm
of clarase was not the optimum amount for hydrolysis.
1949, fasting blood. from AS was collected.
On February 25,
To 50 cmrn aliquots of the
blood. filtrate were added 23, 30 and. 40 cmm of clarase resulting in
blood thiamine values of 3.49, 3.18 and 2.82 mcg per cent, respectively.
Increasing the amount of clarase used was of no value.
Less than 23 cam of clarase was not sufficient.
On January 28,
1949, when 20 and 23 cam of clarase were used, the blood. thiamine
values were 1.46 and 3.06 mcg per cent, respectively.
Use of acid. phosphatase for enzyme hydrolysis - On February 10, 1949,
Dr. Bu.rch suggested the preparation of an acid. phosphatase from human
seminal fluid according to the directions of Dr. Gerhard Schmidt.
This
enzyme had been used for a few determinations by Dr. Birch and 10 cam
of the preparation was sufficient for their experiments.
The seminal
phosphatas? (acid phosphatase) was prepared. according to the directions
of Dr. Gerhard Schmidt (1949).
1
Human seminal fluid was obtained from the laboratory of Dr. Car]. J.
Heller, University of Oregon Medical School, Portland., Oregon.
75
The possible use of another enzyme was encouraging since we had.
been dependent upon the limited supply given us by Dr. 3urch.
The
method of purification of the clarase, furthermore, was not always
successful in producing an active preparation even in the hands of
those who had. prepared. it.
nation
Comparison of the two enzymes by determi
of the concentration of
thiamine in the blood indicated that
10 cmm of acid. phosphatase were as effective as 23 omm of c].arase
(Table 16).
In the few cases in which 23 cmm of acid. phosphatase were
used the values were as high or even slightly higher than those obtained
with 10 cma of
acid. phosphatase.
the larger amount
of
However, the blanks were so high with
enzyme that it was decided. to use the smaller
amount.
Testing the activity of enzymes using cocarboxylase - From the
above
studies, it was 1town that the enzymes were active but it was not
own whether or not they were 100 per cent active.
Dr. J. B. Sumner
(191+9) suggested that the enzyme be tested using sodium pyrophosphate.
In order to make the test more specific a small amount of thiamine
pyrophosph.ate1 was obtained in March, 191+9.
A cocarbolase solution was made up to be
eauiyalent to the
thiamine hydrochloride stock standard as follows:
Thiamine chloride hydrochloride
Thiamine pyrophosphate
Mol. Wt. - 337.276
Mol. Wt. - 1+60.787
50 : 337.276 :: x : 1+60.787
x = 68.3 mg thiamine pyrophosphate which is equivalent to
50 mg of thiamine chloride hydrochloride
1
Appreciation is expressed to Merck and. Company for supplies
thiamine pyrophosphate and thiamine hydrochloride.
of
z1
Dilutions of both the thiamine hydrochloride and. cocarboxylase stock
solutions were
d.e in exactly the same mauner, corresponding to 1 mog
Equivalent amounts of cocarboxylase
per ml of thiamine hydrochloride.
or thiamine hydrochloride standard. solutions were athied to aliquots of
blood supernatants and. these samples were carried through the procedure.
using
The per cent activity of the enzyme in question was determined.
the average corrected readings, e.g.:
corr. read. cocarboxylase
corr. read. thiamine hydrochloride
(100)
% activity of the
enzyme
The results using the above procedure (March 7, 1949) and. 23 crnm
clarase indicated a 102 per cent activity for the clarase.
results
The
using the above procedure and 10 cmm of acid phosphatase
indicated a 99
per cent
activity for the acid. phosphatase.
The
experiment was repeated on March 15, 1949, when 23 omrn of clarase were
found to be 109 per cent active and 10 cmm of acid phosphatase were
These figures seem to indicate that
found to be 109 per cent active.
both enzymes are satisfactory in the quantities used. for the
hydrolysis of thiamine pyrophosphate to free tiiamine.
!iations in. Incubation Procedure
Using longer time for incubation - The procedure as first described
suggested 1 to l- hours for incubation.
On January 18, 1949,
Dr. Bu.rch suggested a 2 hour incubation period with the statement"tbere
may be proteolytic enzymes still
present which tend to promote
hydrolysis to fluorescent materials on long incubation, which increase
the irradiated reading.
The effect of these should be avoided".
77
Table 16
THE DETMIN.TION OF BLOOD THIAMIHE WEBB 2 DIFRER1RF ENZYMES
WERE ADD TO 44LIQUOTS OF BLOOD SUPER1IkTJUT BOM SUBJECT BWO
ite
clarase
19Ls.9
23 cimu
acid phosphatase
23 osm
10 cmm
1h60
k.86
Feb. 22
3.86
3.68
Mar.
1
3.12
Mar. 29
2.5k
2.76
1.57
Feb. 20
3.87
2.58
2.84
3.19
Mar. 31
2. 6L
2.69
2.80
The determination of the concentration of blood thiamine after 1
and 2* hours of incubation are given in Table 17.
In no case did the
values approach those found in normal subjects by D. Burcb.
2
However,
hours of incubation did. not seem to produce any increase in
fluorescence so this amount of time was
throughout
this work.
incubation
nation of blood
satisfactory
It may be that later work will prove 1
hours sufficient incubation
Using higher
considered
to 2
time.
temperature - In macromethods for the d.etermi
thiamine higher incubation temperatures,.k2 to5 degrees
Centigrade have been used. (Friedemaun and Kmieoiak, I9J43, Greenberg and
Rinehart, 19L.5).
The incubation temperature used. in this micromethod.
for the determination of blood thiamine was 37 to 38 degrees Centigrade.
On March 1, 19k9, both clarase and acid phosphatase were used to test
This
the response of both enzymes to increased incubation temperature.
was done by following the usual procedure for the determination of
blood thiamine except for the addition of 10 cmm of acid phosphatase to
half of the blood
supernatant samples and. 23
cmm of c].arase to
the rest
of the blood supernatants and. incubating all of the samples at an
average temperature of 144 degrees Centigrade
for Z- hours.
Table 17
DETEB4INAT ION OF THE OONCETBAT ION OF THIAMINE IN THE BLOOD
AFTER 1* AIW 2 HOURS OF INCUBAT ION
l
Hours of Incubation
Blood
Subject
3te
19149
Apr. 12
BWC
Thiamine
2
1.te
Hours of Incubation
Blood
Subject
Thiamine
moo
mc
19149
2.05
Feb. 22
EWO
3.68
3.86
3.87
1.5
31W
14.147
Apr.
1J4
EWO
3.75
Apr. 21
BWC
3.17
Feb. 25
OAS
3.149
Apr.
5
CS
1.92
Apr. 26
CAS
1.714.
Apr.
The concentration of blood. thiamine (31W) was found to be 3.12 mcg
per cent when 23 cmrn of clarase were used and 2.58 mcg per
cent when
79
10 cmm of acid phosphatase were used.
These values are similar to
those obtained with incubation at 37 to 38 degrees Centigrade.
Variations in the Procedure for Oxidation to Thiochrome and in the
Extraction of the
hiochrome
Use of larger amounts of potassium ferricyanide - In all cases the
solution remained yellow for the entire 15 seconds after the oxidation
mixture was added.
Ohemists,
This has
Inc., 19i47)
to be
been
considered (Association of Vitamin
evidence that sufficient potasiuin
ferricyanide was present.
To confirm this obeervation for the micro-method, one experiment
was performed using an oxidation mixture containing 0.16 per cent
potassium ferricyanide instead of the usual 0.12 per cent of potassium
ferricyanide.
The
usual procedure
was followed for the determination
of blood thiamine for half of the blood samples and to the other half
was added the oxidation mixture containing the higher amount of
potassium ferricyanide.
No significant difference was noted in the
results since a thiamine value of 2.21 meg per cent was obtained with
the usual oxidation mixture and a value of 2.3LI. meg per cent was
obtained for the stronger oxidation
mixture.
Omission of the sodium dihydrogen phosphate - peroxide mixture - Most
of the macro-methods for the determination of blood thiamine
the sodium dihydrogen phosphate-peroxide mixture.
do not use
It was decided to
determine the effect of the omission of this reagent.
On February 26, 19J4.9, the micro-thiamine procedure was
carried
out according to the usual method using only half of the samples.
The
other half of the samples were carried through the same procedure up
To the latter samples were added 0.1 ml of the
to the oxidation step.
oxidation mixture, each tube was tapped for 1
seconds, the sodium
dihydrogen phosphate-peroxide mixtu.re was omitted and 1.0 ml of n-butyl
alcohol was added immediately.
The blood
thiamine value (OAS) following the usual
1.52 meg per cent.
method was
The blood thiamine value (OAS) when the sodium
dihydrogen phosphate-peroxide mixture was omitted was L.29 meg per cent.
This higher
value is not
a valid one because the n-butyl alcohol layer
from these samples was very cloudy.
nor
the addition of a
few crystals of anhydrous sodium sulfate remcved.
this cloudy appearance.
about
The blank was very high, 13.95, instead of
3.80, and all of the
readings were very erratic.
27, i9L.9,
On March 11 and
Neither repeated centrifugation
Dr. Bureb wrote that the sodium
phosphate-peroxide mixture was added to lower the pH
and
stated that
if the solution was too alkaline there would be turbidity in the u-butyl
alcohol layer.
laboratory.
The same observation had been made earlier in this
The chief reasons for the
use of
the sodium phosphate-
peroxide mixture are:
(1) to lower the
blaflk
reading
(2) to avoid turbidity in the n-butyl alcohol layer
(3) to remove the yellow color
Variation in the amount of sodium dihydrogen phosphate-peroxide mixture
used. * In the determination of thiamine in the urine it bad been found
(Mickelsen, Condiff
and Keys, 19145)
that lowering the pH before the
extraction of the thiocbrome aided in the selective absorption of
81
thiochrorne.
ven though it is possible to get only an approximate pH
with the hi
salt concentration present the pH (Table 18) was
determined after the addition of varying amounts of the sodium
The micro-pR beakers were used.
dihydrogen phosphate-peroxide mixture.
for these determinations.
Table 18
TE pH OF OXIDIZED SALES AFTER TI]
ADDITION OF VARTING AMOUNTS OF
SODIUM DIHYDROGEN PRO SPHATE - PEROX IDE BEAGENT
pH
Amount of
(average of det 'us.
2POL1. -
Apr. 1 and 2, 19k9)
Added
9.90
10.00
9.27
8.8k
8.56
100
120
130
ikO
150
On April 1, l9k9, the usual procedure for the determination of
blood thiamine was followed except for the addition of 100 cmrn or 130
cmm of sodium dihydrogen phosphate-peroxide reagent to aliquots of
blood filtrates (BWC) resulting in blood thiamine readings of 2.91 and
2.3k mcg per cent, respectively.
On April 2, 19149, the experiment was
repeated using 100 cmm or 1L0 omm of the sodium dihydrogen phosphate-.
peroxide reagent.
In this case the blood thiamine values were 2.7k
and 3.17 mog per cent, respectively.
A change in the pH at this step
in the procedure was, therefore, of no added value.
Use of iso-butyl alcohol instead of n-butyl alcohol - If the thiamine
was not lost or destroyed and if the thiamine was properly oxidized to
thiochrome it was thought that possibly the n-butyl alcohol was not
Since, in other methods for the
extracting all of the thiochromo.
determination of thiamine (Hennessy
Straub, 191i1, Friedemann and
and. Oereoed.o, 1939,
Kznieciak, 1914.3, Greenberg and Rinehart,
1914.5) iso-butyl alcohol had. been used
t was decided to try iso-butyl
for the extraction of thiochrome
procedure as usual and the thiocbrome
The values obtained. for blood
was taken, carried
concentration of
thiamine
through
the
extracted with iso-butyl alcohol.
thiamine were 5.27 and 5.29 meg per cent.
These were some of the highest values obtained
not
On
alcohol in this micro-procedure.
March 18, 1911.9, non-fasting blood (Ewo)
but the results were
Conner and
under any
circumstances
confirmed on the next day, March 19, when the
in the blood was determined on 5 subjects as
indicated in Table 19.
Table 19
TEE CONCENTRATIOI OF THIAMIEE IN TEE BLOOD OP 5 SUBJECTS AS
DETEBMIEED WHEII THIOOERO
WAS EXTRACTED USING ISO-BUTYL ALCOHOL
!.1y.
BID
BWC
MLW
KEY
GAS
2.83
3.93
1.96
2.03
2.93
83
Reading Samples Sooner after Transfer to pptically Matched Tubes
Some workers (Conner and Straub, 191f1, Friedemann and Kmieciak,
l9L.3) have indicated that the tubes should be read as soon as possible
after the oxidation to thioehronie.
The usual procedure in the
laboratory was to oxidize all of the tubes, which might be as many as
50 or 60, and. then to extract the thiochrome and transfer the alcohol
layer of all of these tubes before reading the samples.
In macro-
thiamine determitious in blood carried out in the nutrition laboratory
little difference had been found in the thiochrome readings whether they
were read immediately or held. at room temperature for a short while and
then read..
However, on March 31, 1914.9, the time before the tubes were read.
was shortened by carrying only 15 tubes through the oxidation,
extraction and transferral of the n-butyl alcohol layer and tben
reading them before continuing with the next group of 15 tubes.
The
values ranged from 2.614 to 3.19 meg per cent which were similar to
those which had. not been read immediately after transfer.
Variation in the Irradiation Procedure
Use of a new irradiation lamp - The drop in readings after irradiation
was not as great as Dr. Bu.rcb had. found.
It was thought possible that
the H-5 lamp was not destroying all of the thioclirome present.
H-5 lamp was obtained.
A new
On May 17, 191+9, half of the blood samples were
irradiated with the irradiation lamp we had been using and the other
half were irradiated with the new H-5 lamp.
irradiated 30 minutes.
All of the samples were
The values for blood thiamine when the
o1dtt
E-5 lamp was used.
was 1.68 meg per cent and with the
w" E-5 lamp
was 1.17 meg per cent.
ven though the irradiation
Variation in the time of irradiation -
lamp seemed adequate it was thought possible that all of the thiocliromo
was not destroyed in 30 mInutes so on February 22, l9Jl9, tubes were
read. after 30 minutes of irradiation, put back and irradiated an
of 60 minutes, and. then read.
additional 30 minutes, or a total time
No consistent difference in. the readings occurred. as a result
again.
of longer irradiation (Table 20).
Table 20
T
1TEOT OF IBRLDIATXNG ThE SA14PL1S FOR 30 AND 60 MINThS
Lverae Corrected Raadins
30 Minutes of
Irrad.iat ion
60 Minutes of
Irradiation
2k.00
23.00
2L$.50
23.00
19.75
22.50
20.00
20.75
19.50
22.50
19.50
22.75
It was suggested that irradiation of the samples for as long as
30 minutes might increase or produce extraneous fluorescence in the
tubes resulting in only a slight final decrease
read after 30
minutes of
before irradiation.
when the tubes
were
irradiation when compared with the reading
Therefore, on April
29, l9L9, aliquots of the same
blood carried through the usual procedure were irradiated. 5, 10, 15, 20,
25 and. 30 mizmtes (Table 21).
me decrease in fluorescence due to
irradiation seemed to come in the first 5 minutes and irradiation for
longer periods was without effect.
Table 21
THE AVEBAGE CORRECTED READING OF SANPIS AFTER BEING IRPADIATED
FOR VARYING LENGTHS OF TI)
Average
Corrected
Reading
Length of
Irradiation
Period.
mm.
8.57
7.97
9.00
6.11
12.02
8.38
5
10
15
20
25
30
Use of New Reagents
All new reagents were obtained and. new solutions made up from these
reagents.
Each new reagent was then checked individa11y by determining
the concentration of blood thiamine using the new reagent and the old
reagent with aliquots of the same blood.
Experiments were performed
using these new reagents on March 17, 21, 23, 29 and. April 2, 1949.
A].].
of the blood thiamine values when the new and the old reagents were used
were still between 2 and 4 meg per cent.
Use of Another Farrand. iicroPhotofluorometer
The Farrand. microphotofluorometer used. during the study belonged
to the Western Regional Project.
Diring the course of this study, the
rrand micro-photofluorometer ordered. by the nutrition research
laboratory arrived..
This afforded an opportunity to road the blood
samples on another instrument.
On March 5, 1949, both micro-
photofluorometers were used. to read. the same blood samples.
No
essential difference in the readings was found between the two
instruments.
The Determination of Standard Ourves
Addition of thiamine hydrochloride to blood supernatants - Experiments
were performed. on April 5, 12, 14, 1, 16 and 21, 1949, in which
standard thiamine solutions of varying concentrations were added to
blood supernatants and these were carried through the procedure.
By
subtracting the value for blood thiamine from the value for blood
thiamine plus added. thiamine hydrochloride the reading for the standard
thiamine hydrochloride itself was obtained.
Details of the above experiments performed on April 5 are described
below:
Twenty-five 50 cmm samples of fasting blood. were delivered. into
6 x 50 mm test tubes containing 225 cunu of 5 per cent trichioroacetic
acid.
These tubes were buzzed immediately, covered with rubber caps
and allowed. to stand 30 minutes.
After the 30 minutes the tubes were
centrifuged at lull speed for 10 minutes and. then 200 cmm of the
supernatant were drawn off into 10 x 75 mm tubes.
The 25 blood samples were divided into five groups and treated as
follows:
Group 1 - Blood samples were carried through the usual procedure;
I.e., 20 omm of 4 M potassium acetate, 23 cmm of clarase
87
(incubation period of 2 hours at 37 to 38 degrees
Centigrade), 100 cmm of oxidation mixture and 100 omm of
sodium dihydrogen phosphate containing 0.015 per
cent hydrogen peroxide were added and the extraction was
carried out with 1 ml of n-butyl alcohol.
55
Group 2 - 5 cmm of standard thiamine hydrochloride solution
containing 1.25 mmog of thiamine hydrochloride in the 5 cmm
were added to each blood supernatant. The rest of the
procedure was the same as described for group one.
Group
3-5
cium of a standard thiamine solution containing 2.50 mmcg
of thiamine hydrochloride in the 5
cmiii were added to each
blood supernatant in the same manner as group two.
Group 4 - 5 cmiii of a standard thiamine solution containing 3.75 mmcg
of thiamine hydrochloride were added. to each blood
supernatant in the same manner as in group two.
of a standard thiamine solution containing 5.00 mmcg
Group 5 - 5 c
of thiamine hydrochloride were added to each blood
supernatant in the same manner as in group two.
In order to be able to express the mmcg of thiamine hydrochloride
added in terms of meg per cent, as we did with the blood values, the
following calculations were made:
according to the formula used, the
5 cmm sample containing 5 mmcg of thiamine would be equivalent to a
blood reading of 13.75 meg per
cent
of thiamine.
For example:
fmmc thiamine hy&rochloride\(corr. read. sampleS) f 100 \
((m). blood) vol. supernatant ) corr. read.
std. ) l000J
total volume 1
I
((0.05)
V64..00\(o\= 13.75 mcg
meg
thiamine
thiamine
J6k.00/(loo0J
275/
So:
1.25 mmcg of thiamine hydrochloride equivalent to 3.4.4 meg % thiamine
6.88
2.50
10.31
3.75
13.75
5.00
The average corrected readl.n.gs for the sarples were:
AVERAGE CORRECTED READING
TUBE CONTENTS
Blood only
Blood + 1.25 nlmcg thiamine
8.83
18.05
Blood + 24.50 mmcg thiamine
Blood + 3.7.5 mmcg thiamine
Blood + 5.00 mmcg thiamine
32.25
14.3.80
When the valt
52.08
for blood thiamine was subtracted from the value
for blood thiamine plus stand&rd thiamine hydrochloride, the results
were:
AMOUBT OF THIAMIiE
READING FOR BLOOD THIAMINE
+ STAI1)&RD THIAMINE HYDROCHLORIDE
- READING FOR BLOOD THIAMINE ALONE
(18.05 - 8.83)
9.22
mmeg
(32.25 * 8.83)
(1+3.80 - 8.83)
34.97
5.00 mmcg
(52.08 * 8.83)
1+3.25
1.25 mmcg
2.50 mmcg
23.1+2
This same procedure was followed for all of the following
experiments also and the results are given in Table 22 and Figure 1+.
Table 22
AVERAGE CORRECTED READINGS OF BLOOD SDPEBNATANT PLUS
THIAMINE HYDROCHLORIDE MINUS READINGS FOR BLOOD SUP] RNATA'NT
Thiamine
Apr, 12
.te (191+9)
Apr. 114
-
4.94.
3.05
4.67
4.22
Hyd.ro-
oride
Air.
Apr. 15
br. 21
Avera
mmcg
0.625
-
1.25
9.22
9.30
9.85
7.36
8.65
8.88
2.50
23.1+2
19.18
17.140
18.88
20.17
19.81
3.75
34.97
35.25
33.90
24.1+5
30.85
31.88
5.00
43.25
48.80
-
42.1+3
43.25
44.4.3
/G(JRE LI. 5 TANDARD CURVES
USING TH BURCH t1ETHOD
BLOOD THIAMINE (NCCr %)
. -,,
70
, a a
... r
4..
r
0. U U
.1
.L.J.
L(...'. .J 4.
CURVE 1: (THIAMINE
65
HYDROCHLORIDE -#-
BLOODS.N)-8LOODS.N'
-- - CURVE 2.
//
(THIAMUvE
/1
HYDROCHLORIDE *
BLOOD) -BLOOD THIAMINE
/
--CURVE 3: TI-I/AN/NE
ss
HYDROCHLORIDE
TREATED AS BLOOD
---CURVE'-I:COCARBOXYL15E
50...
TREATED AS BLOOD
Q
CURVES.(COCARBOXYLASE+
LU
BLOOD)- BLOOD THIAMINE
K
35_
Li
Qz
LU
/
/ /
/
/
/
/
/
/'/
/'/
/'/
14.J
LU
-
//
//
LU
0
/ /
/
...2
/ ..,'
/ /
//
//
30
/.'
/
//
25 -
/
//
//
,'
-
/1
20
25
,/
,%;'
;,
1'
I
0
I
0.625
.1.25
2.50
3.75
frIIlC& TH/Af'IINE HYDROCHLORIDE
'SUPER WA TAN T
5.00
The average corrected reading for blood (BWC) on April 2, 19k9,
was 12.95 or a concentration of thiamine in the blood of 2.7k mcg per
cent.
If this value is read from
gure if, the value of 12.95 would
be equivalent to 1.75 mmcg of thiamine hydrochloride.
Then:
5: 13.75 :: 1.75 : x
x
if.81 mog
thiamine in the blood (Ewe)
Addition of thiamine hydrochloride to blood - When similar dilutions
of thiamine hydrochloride were added to blood itself rather than to the
blood supernatant on April 26 and 28, 191f9, the values were lower than
when the thiamine hydrochloride was added to the blood filtrate as may
be seen in Table 23.
Table 23
TRE AVERAGE CORRECTED READING OF BLOOD (GAS) PLUS
THIAi'1IE HYDROCHLORIDE }4I1US THE RFDING FOR BlOOD
Thiamine Hydrochloride
mmcg
0.625
1.25
2.50
3.75
5.00
Average Corrected Read.ins
2.56
7.Lf6
18.91
21f.k8,
38.30
Determination of a curve using thiamine hydrochloride alone - Solutions
of thiamine hydrochloride alone were carried through the entire
procedure.
The following values were obtained (Table 2/+ and Figure
):
91
Table 24
AVERAGE CORRECTED READING USING THIAMINE HYDROCHLORIIIEI ALONE
CARRIED THROUGH THE ENTIRE PROCEDURE
Average Corrected Beading
Thiamine Hydrochloride
mmcg
0.625
1.25
2.50
3.75
5.00
7.75
15.00
31.13
M.6.35
67.00
Determination of a curve using cocarboxylase alone - Varying concén-.
trations of cocarboxylase equivalent to the above amounts of thiamine
hydrochloride were carried through the same procedure as described on
April 5.
The results are tabulated in Table 25 and Figire k.
Table 25
AVERAGE COBBE (YiED BEADING USING 000AR3OXYLA.SE ALONE
CARRIED THROUGH THE ENTIRE PROCEDURE
Cocarboxylase Equivalent to
the Ibliowing Amounts of
Thiamine Hydrochloride
Average Corrected Beading
mineg
0.625
8.90
1.2.5
2.50
3.75
5.00
33.09
50.96
66.50
92
Addition of cocarbor1ase to blood - Varying concentrations of
cocarboxylase equivalent to the above amounts of thiamine hydrochloride
were added to blood.
The results are given in Table 26 and Figure 14..
Table 26
AVERAGE CORRECTED READING OF BLOOD (cAs) PLUS 000ARBOXYLASE
MIIWS THE READING FOR BLOOD
Cocarboxylase Equivalent to
the Following Amounts of
Thiamine Hydrochloride
mmcg
Average Corrected Reading
0.625
1.25
2.50
7.95
19.15
3.75
5.00
29.85
38.90
As can be seen from Figure £ and as was found in the Priedemaxm
and Kmieciak method, the standard curve if used, must be defined.
It
would seem that the curve resulting from the addition of thiamine
hydrochloride or cooarboxylase to the blood itself and then carrying
these samples of blood plus standard through the entire procedure,
subtracting the value for blood thiamine alone, would be the most valid
curve to use for the determination of blood thiamine.
Determination of Blood Thiamine After
Oral Test Dose
Thiamine
H.ydro chloride
The daily blood samples in the studies reported in this thesis
were taken from subjects in the fasting state.
The possibility that our
blood thiamine values were lower than those reported in the literature
93
was considered because some investigators bad used blood which was
drawn at varying times during the day and, therefore, might have been
influenced by the immediately preceding thiamine intake.
No reports on the effect of an oral dose of thiamine on the
concentration of thiamine in the blood have been found in the litera
ture, although blood thiamine concentrations after intramuscular and
intravenous injections of thiamine have been determined (Bowlands and
Wilkinson, 1938, Ritsert, 1939).
Por this reason the effect of an oral
test dose of thiamine on the concentration of that vitamin in the blood
was studied on April 7, 19k9.
Blood sales were collected from AS and BLD while they were In
the fasting state and at definite time intervals after the ingestion
of 5 mg of thiamine hydrochloride and a lowthiamine breakfast.
Blood
samples were collected at 8 A.L (this was considered zero time) and.
then the 5 mg dose of thiamine hydrochloride was taken.
blood samples were taken at
,
Additional
1, l, 2, 3 and 1+ hours after the test
dose was given.
The usi.m]. procedure for the determination of blood thiamine was
followed except that the blood samles collected on Apiil 7th were
carried through the transfer of the 200 cmm of blood supernatant to
each 10 x 75 mm tube.
At this point the tubes were covered, frozen in
dry ice and kept frozen until the next day, April 8, when the
analyses were completed.
The results are tabulated in Table 27.
914.
Table 27
T
OO1OE1TBATION OF THIAMIEE HI T
ELOOD AER THE A MIEISTBATIO1I OF
A 5 MG ORA.L TEST DOSE OF THIAMIEE HYDROOHLORI
Subject
Tjrne
ELD
OAS
2.59
2.63
2.65
2.85
3.32
3.46
3.13
2.58
2
3.26
2.93
3
2.89
2.30
4
2.88
2.77
0
1
A peak in the blood, thiamine level was observed. j to 1 hour after
the oral ingestion of
5 mg of thiamine
hydrochloride.
The blood
thiamine value after 1 hour represented approximately a 30
increase
over the fasting
level.
per cent
CHAPTER IX
SUMMARY Q WORK WITH
BURGH METHOD
The blood, thiamine values obtained in the nutrition laboratory
were much lower than those obtained by Dr. Burch, values reported in
the literature or results obtained in the nutrition laboratory with
the Priedemairn and Kmieciac method.
The steps in the procedure for the
Burch method were studied in an attempt to find an explanation for these
low values.
Varying the amout of potassium acetate used. in the
procedure produced no significant change in blood thiamine values.
Different brands of trichioroacetic acid. or the use of redistilled
trichioroacetic acid produced no significant change in the blood
thiamine values.
The activity of the enzyme was tested by the determi-
nation of blood. thiamine (1) with and without the use of clarase and
(2) by using cocarboxylase.
The amount of clarase used. was varied and
another enzyme, acid phosphatase, was used in the method.
Both of the
enzymes proved to be active so the explanation for the low blood
thiamine values did not seem to lie in this step in the procedure.
Longer time and higher temperature for incubation were tried without
success.
The oxidation procedure was studied by observing the results
of the use of larger amounts of potassium forricyanide, the omission of
and the use of varying amounts of the sodium dihydrogen phosphateperoxide reagent.
These variations gave no clue to the problem.
Iso-
butyl alcohol proved to be no better as an extractant than xi-butyl
alcohol.
A delay of 30 to 14.5 minutes in reading the tubes after
oxidation to thiochrome had no effect on the blood thiamine reading.
Variation in the time of irradiation or use of another irradiation lamp
or another Farrand rnicro-photofluorometer resulted in no significant
change in blood thiamine values.
When all new reagents were obtained
and. tested indivithially in the procedure, no change in blood thiamine
value was observed.
Standard. curves were determined by .5 different methods C].) addition
of thiamine
hydrochloride
to blood supernatant (2) addition of thiamine
hydrochloride to blood (3) use of thiamine hydrochloride alone (J+) use
of cocarboxylase alone (5) addition of cocarboxylase to blood.
curve (2) or (5) seemed to be the more valid
curves for
Either
use in the
determination of the concentration of thiamine in the blood. since in
these two cases the standard. was subjected to the same manipulations as
blood itself.
A 5 mg oral test dose of thiamine
subjects while in the fasting
hydrochloride was given to 2
state, Blood samples
were collected at
definite time intervals and analyzed for their concentration of thiamine
according to the Burch method.
A peak in blood. thiamine level occurred
to 1 hour after the ingestion of the thiamine
hydrochloride.
The
blood. thiamine value after 1 hour represented approximately a 30 per
cent increase over the fasting level.
97
CHAPTER X
3TURE WORK ON A MICRO-THOD
SUGGESTIONS YOR
BLOOD THIAMflIE
Modifications in the amounts of reagents, time of reading or
incubation and tests of the reagents themselves have been performed
without solving the problem of our low thiamine values as compared with
those values obtained by Dr. Buroli.
It seems as though our problem
does not lie within any of these modifications that have been tested.
The major portion of blood thiamine occurs in the phosphorylated.
form and much of this may be combined with protein forming a complex
(Good.hart and Sinclair, 1939).
There is a strong possibility that
some of the thiamine is lost in the step in which the protein is
precipitated by trichioroacetic acid.
Hennessy (19M7) stated that
"before the elimination of blood protein is brought about, both thiamin
and cocarbor1ase must be separated from any combination with the
protein".
A suggestion for a future experiment would be to delay the
precipitation of the blood protein until after enzymatic hydrolysis had.
been carried out.
A general plan for this experiment would consist of
the following steps:
1.
Deliver 50 cmm samples of blood into 6 x 50 mm test tubes
containing 100 cmm of redistilled water.
Buzz the tubes
immediately.
2.
Add. 10 cmm of 1.0 N hydrochloric acid to produce a pH of about
2 to L4.
3.
Oheck the pH.
Buzz the hemolyzed acidified samples.
Li..
Heat the tubes in a boiling water bath for 1 to 2 minutes.
5.
Cool the tubes and add 20 cmm of potassium acetate to produce
a pH of M..6 to Li.8.
Check the pH.
6.
Add 10 cmm of acid phosphatase to the test tubes.
7.
Cover the tubes with Parafilm and incubate at 37 to 38 degrees
Centigrade for 1* to 2* hours.
8.
Add. 100 omm of 5% triobloroacetic acid.
9.
Buzz the samples thoroughly.
10.
Centrifuge the tubes for 10 minutes.
11.
Remove 200 cmm of the supernatant to 10 x 75 mm tubes.
12.
Add 0.1 ml of oxidation mixture and tap for 15 seconds.
13.
Add 0.1 ml. of sodium dihydrogen phosphate-peroxide reagent.
lLi..
Add. 1.0 ml of n-butyl alcohol.
15.
Buzz the tubes 15 seconds.
16.
Centrifuge the tubes to separate the layers.
17.
Transfer the n-butyl alcohol layer to optically matched
10 x 75 mm tubes.
18.
Carry the standards and blanks through the same procedure as
the blood samples.
19.
Read. bloods, standards and. blanks against a quinine sulfate
standard as described under
20.
the Burch method.
Irradiate all of the tubes 30 minutes, 3
filament of the H-5 lamp.
21.
Reread all of the tubes.
22.
Qalculate the results.
inches
from the
/
The Bu.rch method eliminates the step in which the thiamine is
adsorbed on
calso.
Another suggested experiment is to use this
adsorbent in the method.
Sodium hydroxide could be added to the above
tubes after incubation to bring the pH to 3.0 to 3.5 and. a small amount
of Decalso added.
This mixture could. be shaken, allowed to stand for
a few minutes, shaken again, the s-upernatant removed and the Decalso
washed in the tube several times with redistilled water.
could be removed from the Decalso by adding a
acidified
definite
The thiamine
volume of
25 per cent potassium acetate, mixing, centrifuging and using
the supernatant for oxidation as described above (except for the
omission of the sodium dihydrogen phosphate-peroxide mixture).
These modifications have not been used. before this because they
involve changes in the Burch method rather than variations in the
procedure.
It is to be hoped that a solution may be found soon so
that a method. may be developed that can be used satisfactorily for the
determination of thiamine in small amounts of blood.
BIBLIOGRAPHY
1.
Alexander, B. and Landwehr, G. Studies of Thiamine Metabolism in
Man.
I. Thiamine Balance. The Normal Requirements of
Vitamin i and the Role of Focal Thiamine in Human
Nutrition. J. Olin. Investigation 25: 287-293, 194.6.
2.
Studies of Thiamine
Alexander, B., Landwehr, G. and Mitchell, F.
Metabolism in Man. II. Thiamine and Pyrimid.ine Excretion
with Special Reference to the Relationship Between
Injected and. Excreted Thiamine in Norma]. and Abnormal
Subjects. J. Olin. Investigation 25: 294-303, 194.6.
3.
Archdeacon,J. W. and. Marlin, J. R. The Effect of Thiamine
Depletion and Restoration of Mu.scular Efficiency and
Endurance. J. Nutrition 28: 24.1-2514., 1944.
Li.
Association of Vitamin Chemists, Inc. Methods of Vitamin Assay.
New York, Interecience Publishers, Inc., 194.7.
5.
Atkin, L., Schultz, A. S. and. Prey, C. N. Ultramiorodetermination
of Thiamine by the Fermentation Method. 3. Biol. Ohem.
129: 4.71-4.76, 1939.
6.
Barker, S. B. and Suinmerson, S. W. The Colorimetric Determination
of Lactic Acid, in Biological Material. 3. Biol. Ohem.
138: 535-554., 194.1.
7.
Benson, R. A., Witzberger, 0. M. and Slobody, L. B.
Excretion of Thiamin in Normal Children.
18: 617-620, 194.1.
8.
Benson, R. A., Witzberger, C. M. and. Slobody, L. B. An Evaluation
of the Blood and Urinary Thiamine Determinations in
Vitamin B1 Subnutrition.
J. Pediat. 23: 4.37-445, 194.3.
9.
Benson, H. A., Witzberger, C. L, Slobody, L. 3. and Lewis, L. The
Blood Level of Vitamin i in Healthy Children and its
Relation to the Urinary Thiamine. 3. Pediat. 21: 659-
The Urinary
3. Pediat.
664, 194.2.
10.
Bessey, 0. A., Lowry, 0. H., Brook, M. 3. and Lopez, 3. A.
The
Determination of Vitamin A and. Carotene in Small
Quantities of Blood Serum. 3. Biol. Chem. 166: 177-188,
1946.
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12.
Bueding, B. and. Wortis, H. The Stabilization and. Determination of
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Thirch, H. B,
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16.
Modifications
Cheld.elin, V. H., Bennett, M. J. and Kornberg, H. A.
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25.
A Revaluation of the Methods Described by
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27.
Goodhart, H. S. and Sinclair, H. M. The Estimation of
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Goodhart, H. S. and. Sinclair, H. M. Deficiency of Vitamin
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Gorham, A. T., Abéls, 3. 0. and Robbins, A. L. The Measurement
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The Determination of Free and
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The Determination of Early
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37.
Horwitt, M. K., Liebert, L, Kreisler, 0. and. Wittinan, P.
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APPENDIX
107
APPENDIX I
DIRECTION SHEET GIVEN TO THE ST3BJECTS IN STtJDY II
January 19148
1.
Finger prick blood
samples will be
taken
each day (come to
laboratory at 6:11.5 each morning; 7:45 on Sunday).
2.
Venip'uncture samples will
3.
The subject mast
be taken every 5th day.
eat all fruits, vegetables and milk that are
for him. The amount of
weighed out
bread, butter, meat, sweet
etc. may vary
according to the capacity of each subject.
The subject mast not
eat any foods other than those weighed out
rolls,
Black coffee will
for him at fliealtlmes.
meals, that is, no sugar or
be permitted.
between
cream or milk may be added.
14..
The subject mast not take any vitamin pills, aspirin or anymed.icine.
5.
Each morning a solution of crystalline
ascorbic
acid. dissolved in
water will be given after the blood sa1e has been taken.
6.
Daring the
second and.
third periods (last 20
solution of ascorbic acid will be given
days) an
after the evening meal
(7:00 to 7:30 or as soon as the foods have been
7.
The study will
last
8.
analyzed).
from January 31st through the taking of a
samule on the morning of March let (30 days).
subject mast
additional
be present
at
blood
This means that the
fl meals.
Schedule:
Come to laboratory - 6:4.5 .LM. on week days; 7:14.5 A.M. on Sunday
700 week days, 8:00 Sundays
Breakfast
n
u
"
1:00
12:00
Lunch
"
"
5:30
6:oo
Dinner
108
APPENDIX II
Table 28
INTAKE OF TOTAL AND NON-FAT CALORIES, RATIO OP TOTAL TO NON-FAT CALORIES,
THIAMINE INTAKE IN TERMS OF MCG PER BkY, MOG PER 1000 CALORIES
MCG. PER 1000 NON-FAT QALORIES AND TKE VALUES OR THIAMINE
IN TKE BLOOD DURING A 30 DAT EU'ERIMENTAL PERIOD1
Study I Norma
caloric Intake
Ratio of
FF0 to
Total Oal
Total FF0
]te
19k?
Oct. 18 2195
1288
1168
1464
19 1976
20 24.51
21 2158
Mean
2195
1334.
Oct. 22 2958
23 2089
179?
1521
2379
25 2099
26 2817
14.32
24.
14.16
24.68
1393
1850
1599
Oct. 27 2362
1534.
28
29
30
31
2034.
1263
24.77
14.54
1464
1336
Mean
2048
2082
2201
Nov.
Mean
Mean
1
Thiamine Intake
Per 1000
Per
mcg
y
Cal
mog
Per 1000
NEC
mog
Thiamine
In Blood
mcg
59
59
60
66
61
1198
1033
1558
1029
1205
54.6
930
523
636
477
88k
106k
61
124.3
4.20
73
60
66
66
6
1621
1166
1276
835
1228
776
692
1066
4.90
814.
6o8
296
518
916
k51
788
65
62
59
71
1070
1248
1269
1300
1066
1191
453
698
988
873
888
798
849
9.76
8.84.
4.92
785
835
757
561
820
64.
14.10
64
1 2110
14.77
2 2321
3 1962
4. 1872
5 2051
2063
1325
1276
1280
1262
1324
70
57
6
68
62
64
1159
1106
966
1050
850
1026
See footnote at the end of thiE table.
54.6
614.
512
635
512
54.5
54.9
477
72?
901
4.14.
674.
499
774
9.57
Table 28
Stud.y I Norma (continue&)
Thiamine Intake
Caloric Intake
Ratio of
ThC to
I.te
Total
NC
Total Cal
191+7
Nov.
6 2578
161+2
7 2505
8 2205
9 3035
11+75
Per I.y
meg
614
145].
1281
1003
975
109k
1161
Per 1000
Per 1000
Cal
)IPC
Thiamine
In Blood
meg
meg
acg%
563
511
8814.
321
538
868
760
521
83k
1+78
773
628
800
697
21+72
1521+
59
60
62
65
62
Nov. 11 2582
16714.
65
11+07
64
1051
1125
1+07
12 2187
13 3288
11+ 2505
15 1936
Mean
2500
424
337
6014.
Nov. 16 2313
114.96
10
Mean
1320
1872
1312
1999
1398
1315
1559
14.55
5114.
61
1391+
56
81i4
68
63
781
1039
1+03
59J
417
665
65
741
320
1+95
7.52
7.72
6.12
110
Table 28 (continued)
Study I Barbara
]te
1947
Thiamine Intake
Caloric Intake
Ratio of
Per 1000
Per 1000
NFC to
NF0
Cal
Total Cal Per ]y
NFO
Total
meg
meg
- meg
Oct. 18 2092
19 2164
20 2296
21 2098
Mean
2163
Oct. 22 2986
23 2190
2585
25 1961
26 75
Mean
2487
24.
Oct. 27 2331
28 2141
29 2373
30 280
31 2287
Mean
2302
Nov.
Mean
1565
1332
1395
1744
1.508
1509
1611
1323
1443
1434
237
Mean
Nov.
1662
1543
1293
1852
1639
3 2545
5
22144
6
7
8
9
10
2856
3030
2463
3106
2187
2728
1475
1003
1236
644.2
478
570
62
76
60
66
68
66
1444.
484.
1873
1277
1238
781
1323
855
67
62
59
73
66
65
1093
1431
1177
70
58
63
68
62
64
184.6
11i46
194.9
552
606
63
1 2052
2 2335
4.
1154
1312
1321
1382
1357
1365
1356
134.6
1883
181
1528
18714.
1419
1697
64
59
65
63
66
59
62
60
65
62
14.94
631
288
550
949
1087
735
911
782
1127
828
957
422
696
107l4
1190
1163
1199
1139
980
580
823
4.98
864.
4.71
744
584
4.19
861
679
11314.
510
794.
1411
1373
1193
4.94
74.9
14.53
771
781
557
829
737
484
104.3
36
1176
1239
538
461
940
823
668
496
672
486
558
1111
1282
meg
87'4
4.68
1OO
Thiamine
In Blood
9.93
844
917
737
854
7.12
6 92
111
Table 28
Study I Barbara (contirn2ed.)
Date
1947
Caloric Inta]e
Ratio of
PC to
Total Cal
ILFC
Total
Thiamine Intake
Per 1000
6.12
4.91
Cal
meg
meg
391
549
337
436
412
599
82?
590
616
639
654
380
594
1679
1512
1758
1497
1549
1599
65
66
62
1006
1250
1037
922
990
1041
Nov. 16 2231
1425
64
847
57
56
68
Thiamine
In Blood
Per Iy
2575
2277
3079
2650
2
2571
Nov. 11
12
13
14
15
Mean
Per 1000
N'0
meg
3148
mcg%
112
Table 28 (continued)
Study I Marion
Thiamine Intake
Caloric Intake
Ratio of
I0 to
i.te
1947
Total
NPC
Total Cal
Per 1000
Ca].
meg
meg
9214
1214.7
576
566
578
80k
90k
1211
1164
1281
120?
1216
62
62
59
63
62
1119
1065
90
509
1100
557
62
76
65
67
67
1167
1556
1080
1155
781
1148
414.9
22144
1618
1480
1402
1221
1745
1493
Oct. 27 20814.
1350
28 2023
29 2327
30 2214.7
31 2233
Mean
2179
13311.
65
66
995
1541
1379
1610
1453
1425
59
72
66
66
11511.
Nov.
70
59
66
69
ink
Oct.
18
19
20
21
Mean
1943
1882
2156
190k
1971
Oct. 22 2600
23 1957
2179
25 1884
26 2599
24.
Mean
20714.
1420
1298
1353
1333
1315
2056
13144
214.73
1669
1680
1372
1 2029
2 2203
3 2036
4.
5
Mean
Nov.
Mean
6
7
8
9
10
1939
2771
2235
3002
2213
2539
614.
63
65
14614.
67
61
61
60
66
1600
63
18114.
Per 1000
NYC
mog
Per ]y
Thiamine
In Blood
aog$
915
973
10.85
613
721
1051
770
946
30].
4148
531
787
477
762
496
630
458
565
737
1155
837
880
697
861
9.93
1012
980
1077
893
1015
785
780
9.03
4.59
481
555
724
808
4.31
679
755
1296
1370
1065
1051
1179
1192
524
494
1416
1013
122sf
795
496
511-9
495
477
350
533
476
777
815
776
579
805
750
8.47
113
Table 28
Study I Marion (continued.)
Thiamine Intake
Caloric Intake
Batlo of
N'C to
1911.7
Nov. U 2301
14.67
64.
12 1832
13 2856
1255
1718
1593
Per 1000
Per 1000
meg
meg
meg
383
601
790
15 1956
Mean
2339
1374.
69
60
58
70
1481
64.
882
992
938
1018
81?
929
Nov. 16 2'496
1630
65
1080
14. 2751
54.1
328
370
Thiamine
meg
6.72
54.6
4.18
639
595
4.08
634.
11.33
663
6.32
114
Table 28 (continued.)
Study I Roberta
te
Caloric Intake
Ratio of
NPC to
1I'C
Total Gal
Total
19147
Oct. 18 2136
19 1958
20 2143
21 2202
Mean
2110
Oct. 22 2628
23 2145
1245
1200
1216
1457
1280
58
61
60
72
61
Thiamine Intake
Per 1000
Per D9y
meg
Cal
meg
1088
1103
1252
io6
509
563
584
480
1125
53k
l434
614.
1141
1925
1078
1317
664
1225
57
66
61
Mean
2275
1568
1552
1356
1316
1512
1461
Oct. 27
28
29
30
31
2135
2005
2225
2339
2254
2192
1392
1246
1318
1700
1529
1437
65
62
59
73
68
65
1000
1409
1095
1545
1211
1252
468
1 2220
2 2239
1573
1268
1366
1185
1217
1322
71
57
1324
1021
1125
989
849
1062
596
456
515
553
451
1592
1606
1520
1771
1458
1589
67
58
63
60
66
63
211.
2222
25 2022
26 2356
Mean
Toy.
3 2183
Mean
1787
5 1882
2062
Noy.
6 2372
14.
7
8
9
10
Mean
2762
2418
2947
2211
2542
6.5
614.
63
66
65
64
1409
111-02
1247
1005
1236
1260
897
Thiamine
In Blood
mog$
87k
919
1030
725
887
728
1240
1485
79.5
651
282
550
999
439
718
1131
831
909
792
876
703
492
661
537
572
'
Per 1000
1FC
meg
9.93
8140
10.12
842
805
5114.
835
698
801
594
508
516
341
885
873
820
567
559
8148
504
799
9.02
115
Table 28
Study I Roberta (continued)
Thiamine Intake
Caloric Intake
Ratio of
Per 1000
Ii'C to
]te
Total
1IFO
Total Cal
l9Lf7
12 2052
1 2836
1k 2689
15 2005
1721
1370
1615
1530
1386
21414.9
15214.
57
57
69
63
Nov. 16 21450
1569
614
Nov. 11 266.5
Mean
65
67
Per
y
meg
Oa.l
mog
1114.7
14.30
1078
86k
525
305
352
9146
Per 1000
NYC
meg
666
787
535
618
821
971
1409
592
140k
6140
9142
38k
600
Thiamine
In Blood
acg$
8.28
5.91
116
Table 28 (continued.)
Stu4y I Beesie
Calorie Intake
Ratio of
NFC to
Total
Total Cal
1170
Date
1911.7
Oct. 18 2248
19 1997
1323
1173
1353
59
59
58
1376
1306
63
60
Oct. 22 3007
23 2068
184O
24
2311.9
1314.1
25 2062
26 2383
1291
1593
1508
61
71
57
63
67
20 2351
21 2169
Mean
2191
Mean
23711.
Nov.
Per 1000
Cal
1170
meg
meg
1107
1083
1276
1077
1136
492
837
923
943
783
872
11449
4.82
14.221.
689
1090
1234
685
1176
4621.
63
63
62
71
6s
65
2147
2 2251
3 zo6o
4. 1897
42
5
2099
1537
1259
1235
1284
1328
1329
72
56
6o
68
62
64
1200
1010
963
1066
6 2274.
7 25214.
8 2264
9 2786
1608
1526
71
60
1i449
6li.
1659
1415
1531
60
69
65
1537
1323
1110
936
1251
1231
28 1792
29 2362
30 2351
31 1946
Mean
2137
Mean
64.
Per 1000
Per Day
meg
1403
1122
1473
1675
1267
1388
Oct. 27 2233
Nov.
114.73
Thiamine Intake
3.
10 2.037
Mean
2377
1096
1181
54.2
543
24.97
519
598
28?
788
967
813
956
2430
791
4.91
781
1053
768
906
721
846
9.57
781
802
780
6.92
'1131
1517
914
1168
470
549
1031
incg
504.
659
479
9114.
Thiamine
In Blood
64.5
559
1449
4.67
562
427
493
80
676
524
956
867
766
4.90
688
776
336
614
564.
528
807
884.
7.12
117
Table 28
Study- I Beesie (contiuu.ed.)
Thiamine Intake
Caloric Intake
Ratio of
NTC to
meg
19k7
6k
67
58
851
967
8k2
1k 2327
15 198k
Mean
2220
IL45
1228
1561
13k6
1399
1396
58
88k
71
64
931
895
Nov. 16 2379
1549
65
777
Nov. 11 2259
12 1820
13 2710
Per 1000
Per 1000
Thiamine
meg
meg
mcg%
377
531
311
380
469
7.52
414
589
787
539
657
665
647
327
502
6.72
118
Table 28 (continued)
Study II Tom
Caloric Intake
Ratio of
MFC to
Total Cal
Total
NYC
]te
191i.8
6L40
595
1911
2808
2225
652
538
1008
823
1174
911
2258
2243
1936
4.14J44
2652
2752
2149
3030
2707
2698
64
2300
2950
2938
3518
2658
2873
Feb. 14 5507
15 14586
16 1+895
17 14.466
18 5258
2962
2593
2979
2858
5 5039
6 3621
7 5144
8 4476
Mean
Feb.
1+514.5
9 1+035
10
11
12
13
Mean
5293
14550
6055
14625
4912
Mean
49142
Feb. 19
1+2142
20
21
22
23
14726
Mean
14700
1+707
14786
14632
mog
1421
21449
1
meg
55
59
59
60
59
508
1445
2303
535
398
678
513
57
17140
1+31
56
1914.9
65
58
59
2506
2780
1997
2194
368
551
2973
54
21437
414.3
27814.
61
61
58
2172
2112
2044
2316
2216
4714.
2547
2957
2841
2852
2865
2812
Per 1000
NYC
meg
25141
14.103
Feb.
Cal
1639
3 14308
Mean
Per 1000
Per Iy
66
59
59
56
60
2560
2522
2323
2392
Jan. 31 3891
Feb.
1 14.268
2 3914.5
Thiamine Intake
57
57
58
60
63
60
61
60
61
2014.6
303
1459
1432
1448
1431
1458
1440
1449
2324
514.8
20140
1432
1849
1806
2393
2082
393
381+
500
1+51
Thiamine
In Blood
acg%
9.30
792
815
901
675
1120
861
10.35
757
661
853
790
751
762
12.00
820
780
713
788
777
776
12.27
912
690
651
633
835
744
8.00
119
Table 28
Studi II Tom (continnea)
Date
19k8
Caloric Intake
Ratio of
NFC to
Total Cal
1FC
Total
7eb. 2k k120
25 14474
26 3490
27 4904
28 4?69
Mean
4351
Feb. 29 4989
Thiam
Intake
Per 1000
Per 1000
Thiamine
In Blood,
Per Dajy
Cal
meg
meg
meg
mcg%
1911
1701
1715
1874
1988
1838
k6k
380
491
382
427
762
679
819
633
749_
728
11.18
25444
61
6
60
60
56
59
2806
56
2052
411
731
13.08
2507
2507
2093
2959
2655
24.1?
120
Table 28 (continued)
Study II Ive
Caloric Intake
Ratio of
NFO to
Total Gal
Total
C
te
1948
Jan.
Feb.
1989
11.79
675
8.70
211.140
443
506
381
673
496
784
678
1135
823
23.57
614.
19714.
3
39
63
3036
2290
6149
14082
2238
2537
4151
2911.6
71
5 5508
3113
2233
2898
6 3730
7 5151
8 14639
.27148
57
6o
56
39
14636
2788
61
48148
2692
3348
56
9
10
11
12
Feb. 14
6104
4982
6653
3905
5298
5314.3
15 4657
16 4888
17 5049
18
59
Mean
14.999
19
20
21
22
23
5297
5240
5327
5154
1888
19614
20
2280
841.5
1995
2166
2707
2907
1435
1412
714.1
55
60
56
355
647
910
783
57
22142
1423
7145
2902
2770
2919
2986
2870
2889
511.
2156
2215
1986
2385
14.04
10 00
23147
464
2218
4144
743
800
680
799
818
768
2925
55
65
58
58
2914.1
555
376
380
1005
7.32
29711W
3713
222?
2991
31416
3098
2997
59
6o
59
5?
58
77_2768
5158
acg
553
2 3692
14
Thiamine
In 3lood
8.70
1501
2650
3701
Per 1000
NYC
meg
633
988
838
1109
892
406
623
535
63
Mean
Mean
meg
64
2.3
Feb.
Cal
2372
2682
Mean
Feb.
Per 1000
Per
y
meg
1 14257
3].
Mean
Feb.
Thiamine Intake
3041
59
1972
2023
2154
2048
2228
5L1.3
437
367
1476
1406
1472
8.314
6144
1418
577
653
719
1429
7140
1432
739
121
Table 28
Study II 1ve (continued)
Caric Intake
Thiamine Intake
Batio of
NFC to
.te
Total
NFC
Total Ca].
1948
Feb. 24. 4444.
2529
25 4513
24.58
57
54
26 14.079
4.572
2228
3108
2562
2578
55
60
55
56
Feb. 29 5217
2800
514.
27 514.5
28 k78
Mean
Per Iy
Cal
Per 1000
FC
Thiamine
In Blood
mog
meg
meg
meg%
Per 1000
1773
1261
1879
1909
1867
1738
399
279
70].
4.61
84.3
371
399
382
614
72?
680
2049
393
732
9.30
513
12.00
122
Table 28 (continued)
Studs II Wesley
Thiamine Intake
Caloric Intake
Ratio of
FC to
mcg
Jan. 31 3670
Feb.
1 3558
2 3501
3 1956
Mean
Feb.
Feb.
meg
meg
1522
2278
14.15
65
57
16114.
2614.5
461
669
63
221+3
61+0
61
2015
51+6
2678
2398
2114
2230
2461
2376
64
2106
1883
1835
1369
2298
1898
507
56
13 331
2022
2499
2593
3311
2096
4363
2501+
44. L4.155
6 3570
7 3716
8 Q10
3943
9 3637
10 1+J+35
11 4302
12 5608
Mean
Feb. 14 3891
2222
1960
2516
16 1+134
17 3865
2185
18 4311_ 2525
2282
Mean
3909
15 3284
Feb. 19 3886
20
21
22
23
Mean
Per 1000
2215
367].
5 1+266
Mean
2296
2312
2010
Per 1000
4537
3520
3567
3726
3847
2099
2918
2042
2091
2250
2280
56
59
60
61
60
56
60
59
55
57
1690
1568
2355
2177
1536
1865
444.].
514
368
73
1+81
1+65
354
51+7
388
Thiamine
meg
663
985
803
1179
908
9.00
786
785
868
10.32
611+
9
797
836
627
908
658
11.01
1+01
11.31
752
17011.
438
14.31
767
723
696
778
721
737
11.16
1417
1750
1700
1821
1678
51i.
2228
573
171+9
58
59
60
1392
1509
17.3k
1722
385
395
1061
599
682
722
8.70
61+
57
60
6].
57
58
59
59
423
41+0
416
430
1+23
465
1#1.5
767
123
Table 28
Study II Wesley (continued.)
Caloric
Thiamine Intake
jitake
Ratio of
WPC to
meg
1948
3781
25 3584
26 29514.
27 4162
28 o62
Mean
3709
2248
1996
1794
2585
2338
2192
59
56
61
62
58
59
1532
1196
1333
1354
b. 29 3872
2245
58
Feb. 211.
Per 1000
Per 1000
ThiamiEe
meg
meg
meg $
14.05
68].
10.32
334
599
1+51
71+3
325
5221.
128
1+2
111.29
388
657
1314.0
314.
597
15.32
124
Table 28 (continned)
Stmiy II Don
Caloric Intake
Ratio of
NFC to
NPO
Total Cal
Total
te
1948
Jan. 31 4089
Feb.
1 3629
2
3
924
0
Thiamine Intake
Per 1000
Cal
11TC
meg
meg
meg
429
577
587
78?
595
721
987
985
1408
1025
10.70
448
317
6k
403
585
463
718
581
947
721
947
783
10.70
420
377
550
746
643
871
709
10.85
2436
2121
550
1068
1544
60
58
6o
1756
2093
542
56
15014
59
1474
1308
Mean
2638
Feb.
4 2919
1822
2325
1983
1941
2574
2129
62
55
60
56
62
Mean
5 4255
6 3332
7 3475
8 4168
3630
59
1878
1400
2437
1675
4069
4719
4083
5513
2291
2763
2578
3297
56
59
63
60
1710
1777
2246
2356
Feb.
9
10
11
12
13
Thiamine
In Blood
meg
Per 1000
Per]y
150
421i.
Mean
755
Feb. 14 397?
15 4277
16 4103
1.7 4359
18 421?
Mean
4187
2289
2597
2565
2480
2444
58
61
63
57
247,5
59
Feb. 19 4703
20 4683
21 3993
22 4607
23 3888
Mean
4375
2861
2918
2464
2627
61
62
62
2284
59
60
2631
58
.57
2549
2431
1681
1924
1898
2097
641
568
410
441
2614
2040
2122
2034
1715
2105
556
436
531
442
441
481
145o
502
1114
9.30
936
776
777
852
914
699
861
774
751
800
9.15
125
Table 28
Study II Don (continued)
1te
Caloric Intake
tio of
NYC to
Total Cal
NYC
Total
19kB
368
1+18
71+7
14.16
720
1487
81+6
1+96
58
58
2213
56
3932
2280
Feb. 29 1+547
2617
Per 1000
NYC
meg
713
683
852
603
1698
1608
1692
1626
__2265
Mean
meg
383
61
28
mcg
1+17
266.5
26 31+26
27 1+375
Ca].
141+2
58
56
58
25 3765
Per 1000
Per ]y
1688
2367
2110
1992
Feb. 21+ 1+049
Thiamine Intake
Thiamine
In Blood
acg%
11.01
1k.20
126
Table 28 (eontirnze)
Stnd.y II Keith
Caloric Intake
Ratio o1
NFC to
Total
PC
Total Gal
te
1948
Thiamine
In B10
9.30
14.07
63k
622
455
2868
1212
1930
1187
2057
1597
960
790
1307
923
3321
1949
1188
1908
2938
2261
1949
1149
709
1145
1662
1323
59
59
60
60
57
59
1332
811
538
809
1628
1024
401
416
453
9 2503
1378
1900
zio6
2335
1429
1830
55
993
1096
397
323
164.9
1489
1505
93
1227
374
352
388
64.5
1890
1732
1863
1811
1969
1853
58
1291
1260
398
425
683
56
60
11514.
34.9
1345
44.4
5?
14.22
409
58
1294
4.05
1828
2554
1753
2116
1705
1991
56
66
58
59
62
60
1714
1377
526
355
328
381
348
388
4.
Mean
10
11
12
13
Mean
3394
3369
4024
2498
3158
Feb. 14 3247
15
16
17
18
2964.
3306
3028
3472
3204
Feb. 19 3260
20 3878
21 3014
22 3577
23 274].
Mean
meg
Per 1000
}WC
meg
65
58
57
61
5
6
7
8
Mean
Gal
64.
Mean
Feb.
Per 1000
y
Per
meg
1911
2011
1502
1574
1750
Jan. 31 2978
Feb.
1 3105
2 2609
3 2781
Feb.
Thiamine Intake
3294
56
63
58
57
58
58
990
1364
953
1280
74.0
556
424.
554
450
meg
683
706
759
707
980
767
11.01
721
9.00
577
783
625
670
9.68
727
619
743
722
699
938
539
565
64.5
559
64.9
8.00
12?
Table 28
Study II Keith (continued)
Thiamine Intake
Caloric Intake
Ratio of
NFC to
te
Total
I1'C
Total Ca].
l9Lt8
Per 1000
Per
meg
.y
C
meg
meg
619
566
719
520
61
57
62
62
59
60
1176
378
10144
32k
1166
1063
1332
1447
3087
1900
18k3
1622
2044
1882
1858
116
32k
k15
378
Feb. 29 393k
2205
56
114.7k
375
Feb. 2k
25
26
27
3109
3227
2607
3285
326
Mean
Per 1000
Ca].
Thiamine
In Blood
11.32
626
668
13.93
128
Table 28 (continued)
Stu4 II Ruth
Caloric Intake
Batlo of
FC to
NFC
Total Cal
Total
te
1948
Jan. 31 1461
Feb. 1 1728
501
383
532
370
736
863
5014.
922
1154
1053
1273
804
1041
62
55
6
1448
63
6i
62
66?
730
678
859
545
696
1177
997
61
4. 1566
1081
5
6
7
8
1374.
9 111.89
Mean
2083
1610
2024
1271
1695
Feb. i14. 1932
15 1555
16 1832
17 1375
18 12148
Mean
1588
Feb. 19 1098
20 1817
2]. 1080
22 2011.7
23 1227
Mean
1454.
meg
784
889
745
761
Feb.
10
11
12
13
mog
69
59
68
59
6o
63
1012
Feb.
Cal
444
698
Mean
Mean
Per 1000
Per ]y
648
1206
725
1336
979
1019
1049
937
2 1481
3 1754
1606
2322
1400
2059
1543
1778
Thiamine Intake
1011.2
958
1212
922
1109
70
6].
63
59
63
614.
1314.3
777
719
1003
57
702
1216
675
1301
758
930
64
58
63
67
63
64
62
64
490
762
599
350
421
Per 1000
NYC
meg
636
1150
774
1282
Thiamine
In B10
mog%
8.70
96].
725
647
778
628
1231
802
9.30
723
633
10.00
14.29
644
675
678
4.14
671
779
403
662
673
632
869
982
764
11.00
8.35
424
67].
14.32
811.9
463
675
706
736
4.91
951
708
866
390
4.514.
4.20
1355
582
673
85].
4.16
6514.
652
723
531
860
825
566
14.71
525
129
Table 28
Study II Ruth (continued.)
De.te
Caloric Intake
Batto of
NFC to
Total Oal
NFC
Total
1948
Feb. 24
25
26
27
28
Mean
981
1192
1196
1106
740
626
685
677
663
678
1462
60
860
1212
Feb. 29 2442
94.9
meg
Per 1000
NPC
meg
386
398
490
406
611
660
698
568
376
55)4.
471
618
352
588
Per 1000
y
Per
meg
63
60
70
72
68
67
1915
1573
1399
1666
1261
1663
1
Thiamine Intake
Gal
Thiamine
In Blood
13.93
Values for total Calories and. thiamine were obtained, from the
following references. The nonfat Calories (NFC) were calculated
from the values for protein and carbohydrate obtained from the same
tables.
Bowes, A. and Church, 0. F. Food Values of Portions Commonly Used.
Philadelphia, Philadelphia Ohild Health Society, 311 8.
2d. ed..
Juniper St., 1939.
Bureau of Euwan Nutrition and. Home Economics, U.S.D.A., in cooperation
with the National Beseareh Council. Tables of Food Composition in
Terms of Eleven Nutrients. U.S.D.A. Misc. Pub. No. 572.
New York, Houghton
Chaney, M. S. and. Ahlborn, M. Nutrition. 3d. ed.
Mifflin Co., 194.3.
Cooper, 1. F., Barber, B. N. and. Mitchell, H. Nutrition in Health and.
Philadelphia, J. B. Lippincott co., 194.7.
10th ed..
Disease.
Federal Security Agency, U.S. Public Health Service. Food Value Table
for Calculation of Diet Becord.s.
Nutritional Charts. 12th ed.. Pittsburgh, Pa.,
Heinz, H. .7. Company.
Research Dept., H. .7. Heinz Co., 1946.
Lowe, Belle. Lowe Dietetic File. 2d. ed. Ames, Iowa, Belle Lowe, Iowa
State College, 1929.
New York,
4th ad..
Rose, M. S. A Laboratory Handbook for Dietetics.
Macmillan Co., 1937.
Taylor, C. K. Food. Values in Shares and Weights, New York, Macmillan
Co., 194.2.
130
APPEIThIX III
DIR0T IONS FOR SUBJECTS
A.
Saturday, A..M., January 29
The study will begin officially on
between 6:15 and 6:30 A.M.
the
Each person will report to
laboratory at that time daily.
If all goes well, the experiment
will end. with the completion of analyses on March 12, l9iJ9.
B.
The study will be divided into 2 parts of 3 weeks each.
Period I.
Supplements of crystalline thiamine will be given each
day to bring the total thiamine intake up to the
recommended allowance of the National Research Council.
The basal diet will contain 300 micrograms per 1000
Calories.
Period
II. The basal diet will not
Each person will,
thiamine
The total
for
be supplemented this
therefore, receive 300
period.
of
micrograms
every 1000 Calories consumed.
daily ascorbic acid intake
Supplements of ascorbic acid will
will be 25 mg per day.
be given
daily.
airing the first
three days the supplements may be given at night, but
beginning with
the kth day the supplements will be given in the morning.
0.
The basal diet contains about 1000 Oalories.
No foods (except
coffee without cream and sugar) will be allowed
libitum.
Additions to the diet are planned in units which contain about 500
Calories and. 150 mcg of thiamine.
You may decide during the first
three days, how many of these units you need in addition to the
basal diet, but once you make your decision you are expected to
131
consume the same amount of food. each day.
D.
3].00d. samples will
E.
Urine collections
be taken daily.
for 2Lhour periods
will begin after the first
voiding on the first day and. will include the first voiding on
the following day.
volume, of 2%
You will preserve each collection with io%,
O3 in 2
2 days. You will be given
7.
Do not take
2SO4.
This preservative is
a fresh supply every
stable for
other day.
aspirin, vitamin pills or any medicine during the
course of the
entire
experiment.
(Subject BWC has some dietary restrictions
prescription
and
medication by
and. although her regime will not be the same as the
others, it will be constant throughout the study.)
by
12
.APPENDIX IV
CALIBRTI0N OF THE MICBO-PIPETTRS
Micro-ipettes were made in the laboratory and were calibrated by
delivering the liquid into a graduated pipette and noting the volume
delivered and. consistency of delivery of that pipette.
To be sure that
calibration in this manner was reasonably accurate the consistency and
volume of delivery of the micro-pipettes was determined by weighing the
volume delivered on a micro-balance (the author delivered the samples
as done in the laboratory and. Mr. C. H. Wang of the Chemistry Department
carried. out the weighing on the micro-balance).
The readings by weight
on the micro-balance and calibration by volume as carried out in the
nutrition laboratory were very close so that our method of calibration
Results of the weighings in comparison
could be considered acceptable.
with the calibration by volume are given below:
Volume of A. micro-pipettes
as determined. by delivering
water into a graduated pipette
and reading volume delivered.
50 cmm
Weight of water delivered.
by the same M. micropipettes at 18.2 degrees
Centigrade
i9,Lj99 mg
L8.736 mg
Li.9.597 mg
20 emm
20.L58 mg
20.284 mg
50 cmm
19.875 ing
L.9.592 mg
cmm
23.350 mg
2
23.561+ mg
The 50 cmm pipette for delivery of blood was also checked by
delivery of blood. rather than water and found to deliver the same volume.
133
.APPDIX V
Table 29
VALWS FOR HMA.T0ORIT OR 5 StJBJCTS FOR 52 fl&YS
Sujects
te
BLD
BWC
MLW
HY
GAS
26.50
33.33
35.10
32.21
37.93
35.71
42.37
4.0.00
42.65
38.81
39.29
38.62
4.2.00
-
24.76
37.40
36.85
35.34
33.83
1914.9
Jan. 29
30
Jan. 31
Jafl.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
1
2
3
Li.
5
6
7
39.31.
43.31
36.36
41.10
-
*
144.29
40.59
41.96
42.25
41.48
-
4.3.20
144.14
4.0.27
140.91
4.1.46
41.35
41.12
41.67
34.88
39.43
42.31
-
-
13
14.
38.54.
39.55
15
38.09
36.71
33.86
34.78
4.1.00
16
17
18
19
20
40.89
4.4.114.
41.00
42.47
4.3.33
42.16
43.94
2i
36.56
36.36
22
23
33.90
36.97
214.
35.94.
25
26
27
28
36.48
36.03
35.09
42.34
43.28
42.22
37.04.
37.19
38.27
4.1.16
34.51
38.83
30.89
33e74
34.76
34.48
8
9
10
11
12
38.14.
4.1.78
4.3.20
4.3.10
4.0.94
37.17
40.85
40.18
42.73
36.00
40.63
40.00
40.37
4.2.65
44.40
4.2.28
1i1.l8
32.614
-
39.06
38.69
36.69
36.39
37.63
30.20
39.85
-
4.2.54.
-
42.97
-
38.44
39.65
39.13
39.00
38.80
33.90
42.98
39.76
4.2.86
4.1.60
46.02
40.92
44.38
144.88
4.2.93
4.2.94.
43.25
4.2.35
4.3.89
144.09
4.2.52
39.91
39.02
39.17
38.61
4.2.03
4.2.55
4.3.10
43.92
134
Table 29 (continued.)
Subjects
]te
BLD
BWC
MIJW
QLS
1949
Mar.
Mar.
Mar.
Mar.
2
3
4
34.59
36.41
37.24
37.50
43.22
42.12
43.02
43.06
43.46
42.31
41.00
43.18
Mar.
5
36.73
43.46
40.57
Mar.
Mar.
Mar.
Mar.
Mar.
Mar.
Mar.
Mar.
Mar.
6
7
-
36.36
42.52
43.90
42.64
8
35.17
41.59
9
36.51
43.59
10
11
12
13
36.63
33.95
37.25
1l4
Mar.
Mar.
Mar.
Mar.
Mar.
Mar.
Mar.
Mar.
1
33.81
38.58
37.88
43.98
41.66
42.01
37.46
42.53
39.71
38.91
43.18
43.42
43.44
38.24
42.65
38.22
42.62
40.35
41.77
42.64
39.42
39.87
39.22
40.46
38.87
38.93
38.81
43.13
42.63
42.19
33.82
36.46
40.93
42.86
39.36
40.99
37.69
38.33
41.86
48.28
15
33.46
42.39
44.12
36.51
34.96
43.56
40.38
39.93
16
17
18
19
20
21
22
40.85
40.00
39.71
-
44.29
44.23
34.29
40.30
Jii.61
40.16
43.17
37.28
34.85
36.80
35.34
41.94
39.60
38.98
43.36
35.98
39.39
40.93
43.89
34.81
43.08
38.51
-
36.36
39.69
38.57
45.45
-