A COMPARISON OF THREE METHODS
OF ASSAY FOR THE RIBOPLAVIN
CONTENT OF THREE MIXED DIETS

Them for the Degree of M. S.
MICHIGAN STATE COLLEGE

Annanell Campbell
I944

"THESIS.

I. CO?‘TP"~.DISOI‘I 037' THREE T'WTLIODS OF ASSAY
"POP. THE RIROFLAVIN COT‘ITWTT
OF

TVREE MIXED DIETS

bv

d

Annanell ggnpbell

A THESIS

Submitted to the Graduate School of Michigan
State College of Agriculture and Applied
Science in partial fulfilment of file
requirements for the decree of

L-

VASTWR OF SCITNC

Department of Foods and Nutrition
School of Home Economics

1931+

TH ESIS

f2. “ m: CW 1"“ "-7.? WIT

The writer wishes to expreed her preteful appreciation

to Dr. Thelma Porter, Dr. Unrraret Randolph, Dr. Marraret

I
.4 _..

Ohlson, Dr. W L. Nellmann, Miss‘flilma Brewer and ngu

Petty Pullerd for the materials, help and US;

.‘I

made this study poodiole.

TABLE OF CONTENTS

Introduction . . . . . . . . .A .
Peview of the Literature . . . .

Pt} 7qu? '1“. Of. the n, tI-ldffo o o o o o

Tynerimontal Procedures . . . . . .
DlOlO"l“a—I_ anf o o o o o o 0
Chemical Assay Using the Fluoroscope

Microhiolocical Assay . . . . .

?esults ard Discussion of Dednltd . . .
Chemical Aseav Veinj the Fluoroscone

Nicrohiolocical Erna? . . . . .
Summary and Concludions . . . . . .
Bibliocranhy. . . . . . . . . .

Anpendix

Page

‘J'l

f—J
Q

mini? 147.71g
Thniber 'Title are
I Results of the Bioloaical Assay 21
II Results of the Biological Assay, Calculated

from the Standard Curve 22

III Results of the Fluorometric Ass Y 26
IV Lesults of the Microbiolofiical Assay 27
V Comparison of Values “or Ritoflavin in the

Diets from Hicrobiolonical, Fluorometric

and Yiolosical Results Bl
VI Results of th M obiolocical, FluorOmetric

-e or
and PiolOfi a1 lssays 52

PKATWR

Number litle Pave
I Comparison of the Total Weisht Cairs on

the ExperfirKRTtal Diets 23

II Nicrobiolosical Assay, Diet I Values 28

III Hicrobiolosical Assav, Diet II Values 29

IV Microbiolosical Assay, Diet ITT Values 50

INTRODUCTION

-u.-..e

—— “i—F— .—

 

 

 

 

II’TTRODTTCTIOII

That the animal body needs riboflavin for its most
efficient functioning has been recoenized for many years,
and to riboflavin have been attributed many functions now
known to be performed by other factors of the Vitamin 8 com-
plex. The distinction between riboflavin and the other mem-
bers of the Vitamin B family has been impeded greatly by the
lack of methods for its measurement and extraction.

A short time after fat-soluble A and water-soluble B
vitamins were postulated, experimenters discovered that they
were working with at least two water-soluble factors, one
thermolabile and the other thermostable. The thermostable
portion became known as B2.

Among the early workers who attempted to assay this
material were Chick and Roscoe (1929), and Sherman and his
co-workers (1951-1955) whose efforts led to the establish-
ment of the Bourquin-Sherman rat unit. Those who have worked
to improve this technique are Wasrer, Axelrod, Lipton and
Elvejhem (l9hO), who studied the basal diet; Morgan, Cook and
Davison (1958), who worked with the effect of the kind of
carbohydrate in the riboflavin-depletion diet; Clark,
Lechycka and Cook (lGLO), who studied the valre of the vari-
ous B-complex supplements and who greatly simplified the diet

and clarified the symptoms of ariboflavinosis; Shaw and

Phillips (lth), and Nannerinj, Lipton and Elvejhem (lghl),

6

who determined the effect of fat in riboflavin-deficient
diets; and.Street (l9hl), who has reviewed the rat assay
methods in current use in an effort to standardize and
improve them.

Other animals than rats have been used in the bioloni-
cal assay. Chicks, monkeys and mice were used by Day (195h),
and Jukes (1957) outlined the present assay methods using
chicks. Dons were used as the experimental animals by Street

and Cowgill (1959). In 19h0, {och sufigested usine the larval

stage of development of the beetles Tribolium confusum,

 

dropsophila and sitodrepa in the assay of flours for ribo-

 

 

flavin, and in 19h1 Praenkel and Blewett and Barton-Wright

made improvements in Koch's method with Wribolium confusum.

 

Human studies also have been conducted in an effort to

learn the human requirement.

In vitro experiments have paralleled the biological.
Much of this early work was done in Europe. warburq and
Christian (1955) first isolated crystalline riboflavin, or
"flavine" as they called it. Kuhn and Wanner-Jaureae (1955)
are credited with first notina its fluorescence. The same
authors with Kaltschmitt (195M) studied its distribution in
plants as "1umniflavin" by means of a photometer. Charite
and Khaustov (195M) and Nurthy (1957) measured the flavine
csntcnt of foodstuffs by a colorimetric comparison with
potassium dichromate. Cohen (1955) measured the fluorescence

of unknown solutions with a selenium cell. Weisberq and

7
Levin (1957) compared the fluorescence of the unknown and
standards usina fluorescein as the standard. Von Euler
and idler (l95h). Supplee, Ansbacher and Bender (1955),
and‘Nhitnah, Kunnerth and Kramer (1957) made similar
visual comparisons using lactoflavin as the standard.
Von Euler, Adler and Schlbtzer (l95h) used a carbon arc
lamp and blue alass filters. Josephy and Cohen (both l95h)
used filters also, Josephy comparine his samples with fluo-
rescein, and Cohen with lactoflavin. Hand (1959) used a
uranium mlass cube calibrated with standard riboflavin
solutions, and estimated that with it, riboflavin present
in the amounts of one-tenth to four milligrams per liter of
solution could be detected, whereas the colorimetric pro-
cedure was accurate on solutions containinfi from four to
forty millierams per liter. In 1959, Hodson and Norris
described a method for use with foods by which the ribofla-
vin content was indirectly measured, and Sullivan and
Norris (1959) sought to prevent the interference of impuri-
ties with light absorption readings by reading the samples
on the photometer, reducing them with sodium hyposulfite
and reading again, the difference in readinys beins taken
as the riboflavin value. Supplee, Bender and Jensen (1959)
outlined a similar procedure and checked it by rat assay of
the adsorbates of samples. These methods have since been

modified by Ferrebee (1950) and Najjar (19hl) for use with

8
biolonical fluids. Conner and Straub (l9hl) have simplified
these methods and their procedure is in current use.
Much of the work done on the extraction of flavins from
samples was done by Gybhgy, Kuhn and Wagner-Jauresg (l95h).
Von Euler, Adler and Karrer (l95h) are responsible for the

beginning of work which.showed all flavins to be riboflavin.

Experimentation on the microbiolonical assay of ribo-
flavin has been done flar a large part in the United States,
but a search of the literature indicates that the conception
of the need of microoreanisms for riboflavin is due to
Warburp and Christian (1955), who discovered it in large
mnounts in yeasts, lactic-acid aid acetic acid bacteria, and
believed that it was a cell oxidant for these organisms.
Little was done with this information until Snell and Strong
(1959, 1959) and Krauskopf, Snell and McCoy (1959) experi-
mented to find which of flaese groups of organisms needed
riboflavin for growth. Four of the lactic-acid producers
and one Streptococcus exhibited poor growth in the absence
of riboflavin. Further research produced the method of
Snell and Strong (1959) now used.

Revisions of the method of extracting the samples and
removing of interfering, growth-stimulating substances fol-
lowed, the first beina suggested by Reeney and Strong (19h0),
Scott, Randall and Hessel (l9hl), and Wegner, {e merer and

Frans (19I12). Andrews, Boyd and Terry (1952) found a

9

material present in flour after digestion and extraction
which did not contain riboflavin and which did not stimulate
growth unless it was in the presence of riboflavin.
Bauernfeind, Sotier and Boruff (l9h2) showed that some of the
stimulating materials were fatty acids, salts of fatty acids
and alcohols. Stronm and Carpenter (l9h2) revised the
original method of preparation of samples, but there still is
need for improvement as their results with mixed food samples
varied as much as eight-tenths of a microeram per gram of dry
sample.

Before the microbioloaical method of assay was suneested
the bioloaical assay was used as a standard of comparison for
chemical or fluorometric determination. Since that time, and
due to a feeling that the biological assay is not quantitative,
the fluorometric assay has become the accepted standard.
Whether or not the microbiological assay will replace the
fluorometric assay as a standard procedure will depend on the
development of a more complete means of removiny other growth-

stimulants from the sample.

Emmett and his co-workers (l9h1) have compared the
existins standard procedures and have reported "similar"
results on dried milk samples. Strong and Carpenter (19h2)
consider the results of a l ke comparison of method to give
only a "fair degree of correlation". Andrews (19h5) in his
report of the committee on methods of analysis for 19h2-l9h5

believes the microbiological assay'gives the most accurate

10
analysis of the riboflavin content of foods, the fluorometric
assay to dive values of 87 per cent of the microbiolosical
value, and the biolodical assay to five values of 81 per \
cent of the microbioloeical value in the assay of flours and

cereals.

is far as is known to the author, a comparison of
assay methods on a mixed human diet has never been attempted.
This study has been undertaken for the purpose of comparing

the three standard methods of assay on a mixed human diet.

ilPEfIMENTAL PROCEDURE

ETPTRIMENTAL PROCEDURE

The samples assayed by these three methods were the
three diets fed by Brewer and Ingalls (l9hh)% in the study
of human requirement for riboflavin. The diets were similar
in composition, but fluorometric analysis by Brewer and
Innalls revealed that the three diets varied somewhat in
riboflavin content.

The sampling and preparation of food for analysis was
conducted as follows: as the food prepared for consumption
was weiehed, an additional portion was weished for assay
purposes. The food for each meal was combined into a
single sample, mixed in a'Warinq blendor, acidified to a pH
of h.5 with glacial acetic acid, dried to constant weight
at hOOC., around, placed in amber bottles and stored until
assayed. In this shidy the food samples were mixed in the
original proportions of breakfast, lunch and dinner as calcu-
lated from the moisture loss in the drying of each meal, and

equal amounts of all samples of the same diet mixed and

were carried out in semi-darkness.
The composition of the three diets may be found in the
Appendix in Table I. Calculations of food values were made

by Ties Brewer and Miss Incalls.

% Unpublished data from this lahoratory.

12
PiolOVical Assay

The method of Clark, Lechycka and Cook (l9h0) :as
followed for the biolonical assay, usinn twenty-four day
old albino rats of‘tte Sprajue-Dawley strain. The rats
weighed thirty to forty-five prams when they were placed
on the basal diet. The diet was similar to those recommended
by Sherman, and by Clark, Lechycka and Cook (lQLO), but had
an additional amount of fat as simqested by Shaw and Phillips
(l9hl), and Mannerinq, Lipton and Elvejhem (l9hl). The
amount of fat used was the amount minnested by the latter
aiflfllors.

Basal Diet

Cornstarch 50 %
Alcohol-extracted casein 18
Crisco 26
Osborne and Mendel salt

mixture h
Cod liver oil 2 .
Yeast Supplement 6.5

All of the animals were given 50 micronrams of fiiiamine
hydrochloride daily throughout the experiment. It was planned
to give 150 milligrams of Borden's rice polish factor a day,
but as that company could not supply the rice polish factor,

he amount on hand was fed for the first five days, during
Which time a yeast supplement was prepared for the remainder

of the feedini perio‘. The yeast supplement was the same as

q
:0
c+
”J

7;ed in the microhiolosical assay method of Snell and
Strong (1959), and was inen in amounts equivalent to 150

rilli*na”s of whole yeast a day. The rats ate very little

173

I

of the yeast supplement even when it was given on sugar, so

1 0

it was mixau wzth the hasal diet and baked in a hot oven,
with the heat turned off, for ten minutes. The .swlzpplement
was concentrated so that one milliliter of the solution
mixed with ten crams of the basal diet supplied 6.5 per cent
of whole yeast to the diet.

The rats were placed in individual cages with raised,
larme-mesh screen floors, and given the basal diet and dis~
tilled water, ad libitum. They were weighed twice weekly
until a weight plateau was reached and maintained for a week,
or until they lost weight on two successive weighing days.
This depletion period was seventeen and eiqhteen days in
length. At this time they were separated according to weight,
and placed on the experimental diets with four males and four
females in each of nine dietary proups. Each of the three
human diets was fed in amounts to mapply 2.5, 5.75 and 5.0
microgrm s of riboflavin daily as calculated from the fluoro-
metric analysis. (Table I) Negative and positive control
rats paralleled the experimental rats with fmir males and four
females in each group. Negative controls received no further
supplement. Positive controls were divided into three groups
of eiaht animals and received 2.5, 5.75 and 5.0 microerams of
pure riboflavin daily as a standard solution prepared by
dissolving pure riboflavin (Merck) in 0.02 N acetic acid to

contain 100 micrograms per milliliter, and diluted to contain

10 microirams per milliliter as needed.

The experimental period lasted twenty-eieht days. At
the end of that time the rats were chloroformed and one half
of each group autopsied. All Of the nemative controls were

autopsied.

Chemical Assay Using the Fluoroscope

The chemical assay method of Fodson and Norris (1959)
with modifications by Connor and Straub (l9hl) was followed.
Qarples of the three experimental diets were weiehed in
duplicate on the analytical balance and transferred quanti-
tatively to amber class flasks. Fifty milliliters of 0.0h N
sulfuric acid were added and the flasks were weighed. Food
samples were hydrolyzed on the water bath for one hour, cooled,
and the weimht adjusted to the weight before hydrolysis. Ten
milliliters of a solution containinp 0.01 grams of taka-
diastase and 0.01 prams of papain per milliliter were added,
and the flasks incutated over nieht at 3700. The enzymes
were inactivated by heating the samples on the steam bath,
after which the cooled samples were filtered throuch No. 12
Whatman pleated filter paper. Adsorption columns of
pyridine-treated Florosil were prepared and kept wet until
the samples had filtered. Then a twenty milliliter aliquot
was adsorbed on the Florosil, washed several times with
warm, distilled water, and eluted with thirty milliliters
of twenty per cent pyridine in two per cent acetic acid.

lhc eluate was collected in fifty milliliter volumetric

15

flasks and made to volume. A fifteen milliliter aliquot was
pipetted into twenty-five milliliter volumetric flasks, and
one milliliter of feur per cent potassium permanganate solu-
tion added. This was mixed and allowed to stand for one
minute, then three milliliters of three per cent hydrogen
peroxide were added, the whole mixed and made to volume. A
reagent blank was prepared during this process, starting
with the adsorption column. The standard riboflavin solution
containina 100 micrograms per milliliter was diluted to contain
0.1 micromrams per milliliter. The fluoroscope ( umetron)
was standardized with this solution, corrected with the
reagent blank, and the samples read as per cent of 0.1
micrograms per milliliter.

This procedure was repeated on a second set of samples
except that these were autoclaved for fifteen minutes at
fifteen pounds of pressure in place of the hydrolysis period.

This was done to determine the effect of autoclaving of

samples on the fluorescence of the resulting solutions.

Microbiolonical Assay

The microbioloeical assay for riboflavin was that of
Snell and Stronn (1959), except that the samples were digested
over nidht prior to assay to release protein and carbohydrate-
bound riboflavin.

A pure culture of Lactobacillus caseii was obtained

 

from the Experiment Station, Department of Agricultural

16

Chemistry, Michigan State College, East Lansing, Michigan.
This organism was carried as a stab culture in yeast agar,
and transferred every forty-eiqht hours for eight days, at
which time it was used for the assay.'

The various ingredients were prepared according to
Snell and Stronfi's method (1959) except the cystine and the

yeast supplement. The method for preparing the latter is

from Stronn and Carpenter (lQh2).

Cystine Solution
Cystine was suspended in a small amount of distilled
water and heated gently. Concentrated hydrochloric acid was
added drOpwise until the cystine was in solution. This was
cooled and made to volume with distilled water, and stored
under toluene and chloroform in the refrigerator. One milli-
liter of this solution contained one millinram of cystine.
Snell and StronQ (1959) recommended using cystine hydro-
chloride, but this is not readily soluble in water. The
method of preparation outlined above results in the same
solution, and is more easily prepared.
The medium for fifty assay tubes contains:
50 m1. of photolyzed, NaOH-treated peptone
50 ml. of Cystine solution
10 ml. of yeast supplement
5 grams of anhydrous, C.P. glucose
2.5 ml. of salt solution A
2.5 ml. of salt solution B

These were mixed and the pH adjusted to 6.6 - 6.8 with sodium

hydroxide. after the solution was diluted to 250 milliliters

1?

five milliliters were pipetted into each of fifty standard
bore test tubes. This medium is of double strength to allow
for dilution by the addition of samples. An aliquot of
riboflavin-containing extract was added, and distilled water
to make each tube to a volume of ten milliliters. The tubes
were plugged with cotton and sterilized in the autoclave at
fifteen pounds of pressure for fifteen minutes. When they
had cooled, they were innoculated and incubated.

The innoculum was prepared by the method of Snell and

Stronv (1959), and one drop in a sterile standard four

millimeter innoculating loop was used to seed each tube.

Samples of the three experimental diets were prepared
for assay as follows: 0.0h N sulfuric acid was added so
that each milliliter of solution would contain approximately
0.1 micronram of riboflavin as calculated from the results
of the fluorometric assay. The samples were autoclaved for
fifteen minutes at fifteen pounds of pressure. When they
had cooled, ten milliliters of a solution containing 0.1
mrams of each of takadiastase and papain.were added, and
the samples incubated over nidht. Since riboflavin is
found in foods combined withczereals and protein, and since
all of these diets contained cereals, err and meat, this
digestion period was considered necessary. Enzyme action
was stopped by heatinm the samples on the steam bath for
five minutes, and the pH adjusted to 6.6 - 6.8 with N NaCH

solution. After filtering throuah No. 12 W atman pleated

73

r—l
.J-

filter paper, the samples were restored to their original
volume with distilled water. Aliquots then were pipetted
into tubes containing the basal medium, made to a volume of
ten milliliters, and sterilized in the autoclave. Nine sets
of triplicate tubes were prepared, and each of the three
diets was samples in amounts fiving an estimated 0.05, 0.15
and 0.25 micrograms of riboflavin. With each assay, dupli-
cate standard tubes were set up containing 0.0, 0.05, 0.075,
0.1, 0.15, 0.2, 0.5 and 0.5 micrograms of pure riboflavin

Der tube. All of the tubes were innoculated as described

L

0
above, and incubated for 2k, AB and 72 hours at 57 c.

(Table III).

When.the incubation time was finished, the oantents of
the tubes were transferred to Erlenmeyer flasks, the tubes
washed with ten to twenty milliliters of distilled water, and
titrated with 0.1 N. sodium hydroxide (approkimately) to a
pH of 7.0, using Brom Thymol flue as an indicator and a color

comparison flask. The indicator was added to the flasks

before the sarples were transferred.

RESULTS AND DISCUSSION

mjguTTS Al-ID DIS cvssmw

The results of the biological assay of the three
experimental diets are recorded in Tables I and II. A standard
curve was constructed from the total averane weicht gains of
the control animals, but the rise of this curve was not great
enough to allow for calculating the riboflavin content of the
experimental diets from the gains in weight made by the
animals on these diets. It was not considered logical to
extend the st€.dard curve because all of the weirht gains
of the animals on the experimental diets were above the
hishest weieht nail on a pure riboflavin supplement. Thus
the experimental diets seemed to contain much more riboflavin
than was found by fluorometric assay. It is unlikely that the
fluorometric analysis was this inaccurate, and the discrepancy
in weight mains must be explained as due to other substances
in the experimental diets which were erowth-promotind in the
rat, or to an inadequacy in the basal diet. Since the rise
of the standard curve was less than that reported in the liter-
ature, the latter seems the more likely explanation although
this same diet has been used successfully by Shaw and Phillips
(l9hl) and by Mannering, Lipton and Elvejhem (lle). However
even if a standard curve of the usual rise were used, the
weight dains of the animals on the cxperimental diets far
exceed those of control animals except on the hinhest concen-

tration of riboflavin. The smallest tain in_weiqht on the

20
experimental diets, the 2.5 microeram level of Diet Ill,
would then correspond to the hiehest gain in weight on the
control diet.
Plate I shows the total average_nains in weifiht for

each group. It will be noted that the van

Ho
(3"
O
H.)
H
S13
<:
H.
:5

each diet were well separated for each amount of r
fed.

0n autopsy five of the negative control animals had
yellow edded livers, and all had mottled livers. There
was no body fat on these animals, and they were less mature
sexually than any of the other groups; the testes in the
males were smaller and there was no follicular development
in the females. The three groups of positive controls were
much the same, the amount of body fat corresponding to the
riboflavin intake. All of the positive controls had
mottled livers, but none showed yellowing. Testes in male
rats were larger, and the ovaries were faintly pink. The
animals on the three experimental diets were comparab e.
These rats also had stores of body fat corresponding to the
supplement intake. There were no yellow livers, and mott-
ling appeared only on the lowest amount of diet fed. Testes
were correspondinaly larder, and all of the females on the
five microsram level of riboflavin intake in the diets
showed mature follicles. The external appearance of all of
the animals was much the same, except that the negative

contrel rats had less hair and this was matted with oil.

TKPLW I

Results of the Bioloeical A

ssay

 

 

;”amma of amount of diet fed Total Averaxe Veirht
:ribofla- r*ains on the
Vin/brag 4L‘f‘xperimertal Diets

 

 

damra riboflavin

 

—'vv'-“’- v—u.

 

£93318 Of' riboflavin

 

..l

 

 

 

 

 

 

 

 

 

 

 

 

 

Con-
trols

 

 

H.2.5" 5.75 I 5.0 ‘ 275l55.75 l 5.0"
‘**.. .f» . ‘ l-c ‘; r o r. ‘I 4‘ ..
F ,‘PHLS’?::Ex era i, :TQYNJ-:TZE§_.rsIPam° __$
. 7 . “—*‘
I 1.51% .1.65 2.5 5.5 17.5 121.0 E 22.0
L ---_-_-___. ' ....- ,_____.-1.- --_.._._......'....__._.a_..._,
T-v- 7’ .1 QC Q 2 '7 ( i 9 D
‘..'_.l_. l.,)*L J 0 i2...) /.7 1700 1:900 2a.-..)
III .85 '1.b 2.05 2.7 15.0 18.0 g 25.0
t
a
_-. --..-_m__..._--_n_4__._. -- 1 .4

 

 

?
;
I
1 f '

 

 

 

\'
c I
'\

.7 fi—

 

 

 

Values calculated from brewer and Infalls (lth)

Calculated

 

'j
l ‘°
(D
ct

lfaldies

of the

Piolosical Assay
from the

Standard

Curve

 

 

calculated from Standard Curve

 

 

 

 

 

 

 

 

 

 

 

165 2.57

(“edema of riboflavin fed Averafze
2. r; 2 {7.3 | I; .0 earm‘ea/fgm.
per gamma/V per !gamma/4;er namma?
cent pram cent nrmn_lcent erem
' I-II'. s
x I, i
255 b.13 210 5.56 165 2.66 5.h
U

 

III

 

 

 

 

 

2 L

’1. . L+

165

 

 

 

 

 

TOTAL AVERAGE WEIGHT GAINS 0F ANIMALS IN GFAIviS

 

 

 

PLATE I
TOTAL AVERAGE WEIGHT GAINS

THE EiPeHIIsNTAL DIETS

CONTROL ANIIV'ALS DIET I ANIIx'ALS

 

\
\\

 

 

 

 

 

 

 

( r‘-\
10 .30 (JOE
m.- ' .
I are In Days

DIET II ANIE’TALS DIET III ANIIV'ALS

 

 

 

 

 

2h

These results are not quantitative, but they do corro-

borate the findings of Shaw and Phillips (l9hl) with reward

x)

cf-

0 the effect of ariboflavinosis on the reproductive organs.
They would seem to indicate that riboflavin is intimately
concerned in fat metabolism and storave

, as Nannering,

Lipton and Elvejhem (lOLl) affirmed.

Chemical Assay Using the Fluoroscope
There were two sets of samples fo- this assay, one was
hydrolyzed to release the riboflavin and the other was auto-
claved. The results are found in Table III. Autoclavinq of
samples in place of hydrolyzind them had little effect on
the results of the fluorometric assay. The difference was
slidht in all but Diet III, in which autoclaved samples have

readinss of 117 per cent of the hydrolyzed samples. Since
autoclavind apparently made only a small difference, the
values of the hydrolyzed samples were taken as a basis of

comparison of fluorometric values with the values of the

microbiolonical method.

Licrobiolonical Assay
Diet samples assayed by the microbiolonical method of
Snell and Strong (1059) and Strong and Carpenter (19h2)
indicated that the twenty-four hour incubation period was
inadequate and gave misleadins results. (Table IV) Forty-
eiqht and seventy-two hours of incubation have results more

nearly like the standard curves, and compared more closely

25

with the fluorometric assay. The correlation also was
closer between the forty-einht hour standards and safiples,
than between sawples w ich had been incubated for seventy-
two hours. Stronn and Carpenter (19h2) believe that the
seventy-two hour incubation period gives the most accurate
results, but as this did not appear to be true of these
samples, all comparisons were made with the riboflavin
values of samples which had been incubated for forty-eirht
hours. (Table V).

Standard curves were constructed for each of the incuba-
tion periods, and the riboflavin values of the samples were

calculated from them. (Plates II, III, IV).

The results of the three methods of assay are compiled
in Tables V and VI. If the microbiological assay results are
taken as the true riboflavin values of the samples, the
fluorometric assay values are thirty-four to one hundred and
five per cent hicher, and the biological assay values would
be two hundred and thirty-one to three hundred and sixty-
seven per cent himher.

The estimated riboflavin values calculated from the
microbiological assay of each of the three diets indicate
that there is little basis for comparing the three methods
of assay when a nood mixture is tested. The present methods
of ass(y apparently are not accurate enough for use when a

larce number of foodstuffs are present.

TWDLE III

Pesults of Fluorometric Essay

 

 

I Is lP‘ht

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

l
1
:Diet of ‘ Lumetron readincs Gamma Riboflavin/ Diflbr—
sample ! gram ence
j1‘95"“treated Autoclaved Vntreatethutoclavefiler
Tent
I 5 52 55 1.6 1.65 +3.1
. II 5 28 2,.5 1.h 1.575 -1.8
L
(
L:II s 58 L5.5 1.9 2.225 +17.o
I _.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0H. 00H. .. 0e.0 00.5 00.0 0m.0
NH. NH. .. 00.0 00.0 00.: 0H.0 0.HHH 0 HRH 0.0a 0.0 0H.: 0.0
00. 00. 0a. 00.: 0m.: 0m.0 00.0 0.0a 0.0 0.: 0.0
we. 00H. -- 00.0 00.0 00.0 0m.0 0.0 0.0 00.0 m.0
000. 0H. -- 0H.0 00.0 0m.: 0H.0 p.00 0 He 0.s m.s 0.0 0H.0
000. 00. ms. 00.: 00.: 0m.0 00.0 m.0 s.0 0.0 0H.0
00H. 00H. .. 0H.» 00.0 0H.0 0m.0 0.0 0.: 0s.m 0N0.0
000. 00a. -- 0H.0 0s.0 00.: 0H.0 0.m0 0 H H.0 0.: 0.m 00.0
0H. 00.: 0m.: 0:.0m 00.0 m.0 0.0 0m.m 00.0
4N .HW .HW .MW.J mEEwm .HE mfidpmi .HW .HW .HE 095p
1 . (IL m m: m 1 a 1 - m w : sew magma
emmmwcmmm memp mpzom m manfiwm :ommm oHQEwmpoHQ mpzom .ll MQQSp
ueuwHSono mHQEwm Cofipwapfip QEwHwonfip. z :0. mo Coapwnpap pswpcwpm
EH Qw>wakonfim CH @005 mowz “bpmfifipmmfi p3uam3. CH pom: momz

 

hamma HavamoaoHDOHOHE 03p mo mpHSmea

>H mam<e

 

. 'T'T.“
I

’- l
1
Jul...

it.“

T)
1

3.01"“

7r.
.4

s for

.“T’U’Q

h
‘ .1

Standard

, .
Wits

fT‘l ‘ OW

J, ‘1

1'3 0 T1

q-n

l

Plotten

{-

93

Val?

rOTJT’S

 

 

 

  

 

 

 

 

 

 

 

 

 

 

 

 

 

h h“ .5
I [J pair.
- r v -1 - r - 5
fi ((11; 4.. o
.
_
a
7 n no y-l|b)~(x.l+| l ..
_
M
w
1 I.) I ,_-. . I H
_ W
, _ w
l a. M r 1 I a
_
_ 2
.- ii! {all}: , -- til ,5
_ _ .
4
g
u
T. l l i 11.. I . _ . ill . M
_ a. _ m
_ i _ _ _
T a in! i _ m _
a :i5:1£-i|?-li!: 1- . E0
. ,. _ i .
-_ H i
A. i .- I ll. II." J
I I». .1 I. I In)! 1:! llell. I! 11'. .___ w
_ _ Ii . a
J." I IJ IJJAI lllllll i
w . . — . . u
.l l 1 . -;u — (i —1 a, w LA]...—
“ . 9 j
i _ __
.. i1 1 1 l lii-!ll-+ - -Ill-a P T .-
.-, u .m a
_ - ll Iv -- " 'Il'. '0
a _ _
p .- h a —. —. III d - .~ L I .
9H ..m Qua PD 4 2
1 l
ch+wh+09 Cfi 1mm; ECG? V H.O %0 .Hm

“boflafin

.1.
".1.

7—
\
A

we of

VDW

q
K] r] 1,"

logical

in

-v-
o.-

l
icrob

T‘

fw- 9—.
0

ML;
ith

,fi
0

for F

A

(3c
'.u

“VFW-

A
II

andard
Liet

L

0+

NJ

hours

cu

J .
.

L24 hours

 

 

 

 

 

 

 

 

 

v.1 Iii! -11. L.O~Uhd
i
i i
a l- 1 . - 02
a ’ M a f . . 0
. _ _ ._ . .
_ J 4" "l’l'lv‘..- 'r’-l'l'.'"'l
_ _ / IA H X _
I - . . i 1 EL
|!"l

 

 

  

 

 

 

 

 

 

’------y——-.—
.
.

C
E
4 1
2

90 ..
l l

coaeaape. CH 0002 mom; 2 H.0 mo .Ha

r...

S S S
a. r r
A. 0 n.1,
L“ .n .n

L;

0g

4?
L.
1

 

rioloc
Tron

‘

é

-. lCT‘O

‘u

wit.
Plotted

7 1
.alxcs

v

 

”rrvcs for
III

1

‘ict

T
M

. .h _—-—.—-1
E
O V

      

7 u

fl— *.

 

lollill..I ....II.|I lull I. . u I III.- I. . . . .l . A
.
— .

“l|l"¥|' ll' l'll‘|"‘ll

 

 

 

_/ ..

 

 

 

 

 

 

_ ,
-... -.e../...:. .. .. -.
-/ . 1
0p

4.

 

C
1

coaumLu-. Cw Emu; Wow? V H.O %0 .H;

o I...

O
-

lav:

D

.s

fibo

of

TRWLE V

Comparison of Values for Riboflavin

in Diets from

hicrobiolodical, Fluorometric and Biolonical

 

 

 

 

 

 

 

 

Results
Microbiolocical Fluorometric fioloeical
Diet assay value assay value assay value
Gamma/gram Gamma/dram Gamma/tram
I O V 1 60 2 M
.7,5 1.- ,oe
| II 0.89 l.hO 2.8
I
; i
{111 1.M1 1.00 3.66

 

 

 

 

 

TABLE VI

Results of :

 

 

 

 

 

Microbiolonical, Fluorometric and Biological
Assays
Riboflavin
calculated Microbiologi- Fluorometric Biolodical
Diet from micro- cal assaxa assayfi assayu
biological
assay
I 0.775 100 % 205 % 067 a
II 0.89 100 156 551
III 1.01 100 15h 55h

 

 

 

 

 

In per cent of Microbiological assay results.

 

SUI-SHAPE AND CC.‘ICLUSIONS

“VITTWTV ANI3(XVTCLTSICWK3

‘ hen albino rets, which had been depleted of their
riboflavin stores as indicated by'weight plateaus, were fed
the three experimental nixed human diets, their weight gains
far exceeded those of “at: on the basal diet plus pure ribo-
flavin. It is felt that growth-stimulating substances other
than riboflavin were present in the experimental diets
which prevented an accurate determination of the riboflavin
value of the diets, and also that the basal diet did not
permit normal growth and development.

Autopsy revealed that the animals on the experimental
diets had better developed reproductive orsans than the
control animals, and the positive controls showed better
development than the nesative controls. The amount of body
fat was directly related to the amount of riboflavin fed,
but all of the animals given the eyperimental diets had
larger fat deposits than the control animals. Most of the
negative controls had yellow-edged livers, and all had
mottled live-s, as did all of the positive controls and the

experimental animals fed the smallest amounts of the three

diets.

Autoclavinn the food samples before determining the
riboflavin value fluorometrically nave values of -l.79 to

+ 17.0 per cent of the hydrolized samples. From these

351;-
results it is indicated that the substances causina inter—
ference with readinss of fluorescence are not removed by
autoclavina.

A forty-eight hour incubation i.eriod for food samples
by the microbiological assay resulted in values for ribo-
flavin which were more closely related to the standard

curve and to the fluorometric assay values than the results

obtained by seventy-two hours of incubation.

The results of the fluorometric assay were thirty-four
to one hundred and five per cent hiiher than the microbiolog-
ical assay values, and the biolodical assay values were two
hundred and thirtvaone to three hundred and sixty-seven
per cert hicher. Since it is unlikelv that any of the
methods is as inaccurate as this comparison would indicate,

a detailed study of the materials which cause variation in
fluorescence and a means of removing grthh-stimulants for
the biological and microbiolosical assays are necessary
before any comparison of these methods of assay of a mixed
diet for riboflavin can be useful. Due to a large number

of unknowns in a mixed diet, and to innorance of the manner
in.which they may operate as stimulants or interferents,

the present assay methods do not hive comparable results.

BIBLIOGFAPHY

BIBLIOGRAPHY

Andrews, John S., Boyd, Harold U., and Terrv, Dc vid E. lQLQ.
Riboflavin Analvsis of Cereals; Application of the
Ficrobiolodical Method. Ind. Ens. Chem., Anal. Ed. 1“:
271—27h. '

Andrews, Tohn 8.19‘2 Report of the 10J2-‘QL5 Methods of
Analvsis Subcomrittee on Riboflavin Assav, I. Cereal
Chem. 20:615- 625.

Arnold, A., Linsuis, S.T., and Creene, D.J. lth. Ribo-
flavin Determination by the Microbiolocical Method.
Food Res. ozas-La.

Barton -WrL dht, E. l9lc'l. Flour and the Growth of Tribolium.
Nature 118:565- 5ob.

Bauernfeind, J.C., Soticr, A. L., and Boruff, C.S. l9h2.
Growth Stimulants in the Necrobiolo ical Assav for
Bi boflavin and Pantothenic Acid. Ind. Ens. Chem. Anal.

Ed. 1A.666- 669.

Brewer, w11 ma D. and Ingalls, Ruth L. 19th. (Unpublished
data).

Charite, A.J. and Khaustov, N.W. 195A. The Estimation of
Flavincs in Animal Ti sues. C.?. Acad. Sci. 5:388-790.
(Nutri. Abs. and Rev. h:505, 195h_1955)

Chick, a. and Roscoe, M. H. 1928. The Effect Upon Young
Rats of Vitamin B Deficiency'and a Method for the
Piolocical Assay of Vitamin B2. Biochcm. Jr. 22:790-98.

Clark, M.F., Iechvcka, M. and Cook, C.A.19LO.Biolonical
Assav of Riboflavin. Jr. Nutrition 20:153-lhh.
Cohen, F.H. 195M. A Simple Fluorescope Especiallv fo or the
Assav of Vitamin B2. acta. brev. neerland. [::b6- h7.

(Nutri. Abs. and Rev. h:505, 195h-1955)

Cohen, F.H. 1955. A Simple AppAratus for Objective
Fluorescence Measurement by Means of a Selenium Cell.
Rec. Trav. chim. Pavs-Bas 5h:l§3. (Nutri. Abs. and
Rev. 5:0t9, 1955-1956)

Connor, R.T. and Straub, G.J. lth. Combined Determination
of Riboflavin in Food Products. Ind. Eng. Chem., Anal.
so. 15:585-388.

’4
l~

Dav, Paul I. 197h. i"4::’r_.r:-1.ctabilitv of Vitamin C (B )_from
Yeast bv Various AcetonJfiater and Methvl-Alcohol-Water
Mixtures. Jr. Amer. Chem. Soc. 5A:h52-HSM.

Dav, Paul L. 193’ Vitamin C De eficiencv. Amer. Jr. Public
Health. 2A:603- 6OQ

Emmett, A.D., Bird, 0.9. Brown, R.A., Peacock, 9., and
Vandenbelt, J.M. 19'11 ihe Determination of Ribo-

flavin. A Comparison of Bioassay, Nicrobiolo ic cal and
Fluorometric. Ind. Ens. Chem., aral. El. 13:219-212.

Euler B.v., and Adler, E. 195h. Flavines in Animal
i‘sues. Ztsohr. F. thsiol. Chem. 225:105-112. (Nutri.
1s and Bev. h:41, 195h-1955).

3‘.

A

Euler, H. v., Karrer, P., and Adler, E. 195k. Flevines and
Ark. Kemi, Min. Cool. 11B : No. 55, o. 6. (Nut ri.

Q90 , 1
he. and Rev. t:286, 195A-1935).
Vuler, H. v., and Adle E., and schldtzer, A. 193A.

Distribution of Bound and Free Flavines in Plants.
7tschr. f. phvsiol. Chem. 226:87-9h. (Nutri. Abs. and
Rev A: 509, 195A-l955)o

Feenev, R.E. and Stron", F.N. 19MO. The Nicrobiolosical
Determination of Riboflavin. Jr. Biol. Chem. 1;}:
P POC . XK‘CT.

Ferre ebee, Joseph W. 19LO. The Urinary Excretion of Ribo-
flavin. Fluorometric Methois for its Estimation. Jr.
Clin. Invest. 19:251-260.

Fraenkel, C. and Blewett, M. l9hl. Deficiency of White
Flour in PLiboflavin (Tested with the Flour Beetle,
(Pribolium.cordhmrun). 'Nature llfff7l6-7l7.

 

CySrgy, P., Kuhn, R., and Wacner-Jaureej, T. M95 PDGDHPR-
tion of Vitamin B2 Concentrates. Ztachr. f. phvsiol.
Chem. 223:27-35. (Nutri. Abs. aha Rev. lm:5 193M-

1955).

Gyfirev, P., Kuhn, R., add We‘ner-Taure"s, T. 195h. Flavines
and Flavoproteins as Vitamin B2“ Ztschr. f. phvs iol.
Chem. 225:2h1-2hh. (Nutri. Abs. and Rev. h:286, 193}-

1955).
Hand, David B. 1959. Determination of Riboflavin in Milk bv
Photoelectric Fluoroescence Measurements. Ind. Eng.

Chem., Anal. Ed. 11:506-509.

«N
—J

Hodson, A.Z. and Norris, L.C. 1959. A Fluorometric Method
of Determininh the Riboflavin Content of Foodstuffs.
Jr. Biol. Chem. 151:621-630.

Josephy, B. 195L. The Quantatitive Estimation of Vitamin
B by Means of Fluorescence. Acta brev. neerland.
h-hP-L9. (Nutri. Ahs. and Rev.‘h:9h9, 193h-1955)o

Jukes, T.H. 1967. The Bioloeical Assay of Lactoflavin with
Chicks. Jr. Nutri. 1hz225-255.

Koch, A. lQHO. Growth Substances in Yeast. Naturwissene
schaften 28:2h-27. (Nutri. Abs. and Rev. 11:5oh, 19h2-
19MB).

Krauskopf, E.J., Snell, E.E. and McCoy, E. 1939. Growth
Factors for Bacteria II. A Survey of the Pantothenic
1f

finid and Riboflavin Requirements of uqrious Groups of
Bacteria. Enzymolomia 7:527-550.

Kuhn, R. and Waener-Jaurepq, T. 1955. Flavines isolated
from E3: White and kilk. Ber. d. deutsch. Chem.
@esellsch. 66:?577-1582, 1952. (Nut. Abs. and Rev.
5:992, 1955-105h).

Kuhn, R., Kaltschmitt, H. and Waéner-Jaureefi, T. 195M.
The Distribution of Flavines in Plants. Ber. deutsch
Chem. Ges. 67:1u52-1L57. (Nutri. Abs. and Rev. h:509,

1954-1955)-

Mannerine, G.J., Linton, M.A. and Elvefihem, C.A. 19h1.
The Relation of Dietary Fat to Riboflavin Requirement
of Growing Rats. Proc. Soc. Exptl. biol. Med. L6:100-
10h.

and Davison, H.G. 1958. Vitamin

Morgan, A.F., Cook, 9.3.
ffected by Dietary Carbohydrate. Jr.

Deficiencies as A
Nutri. 15:27-95-

Murthy, }.N. 1957._ A Chemical Method for the Estimation
of Flavine in Foodstuffs. Ind. Jr. Med. Res. 2h:1085~
1092. (Nutri. Abs. and Rev. 7:550, 1957-1958).

Najjar, Victor A. 19L1. lhe Fluorometric Determination of
Riboflavin in Urine and Other Biolovical materials.
Jr. 5101. Chem. 1h1:555-56u.

Peterson, W.J., Prady, D.E. and Shaw, A.O. 19h}. The
Fluorometric Determination of Riboflavin in Pork
Products. Ind. Ene. Chem., Anal. Ed. 15:65h-656.

gohumacher, A.E. and Reuser, C.E. 19h0. “iboflavin Content
of Yeasts Determined Photometrically and Biologically.
Ind. Eng. Chem., Anal. Ed. 12:205-20h.

Scott, M.L., Randall, F.E., and Hessel, F.H. 19Ll. A
Modification of the Snell and Strong MicrobiolOfical
Method for Determining Riboflavin. Jr. biol. Chem.
lh1:525-526. (Letters to the Editor).

Shaw, J.H. and Phillips, P.A. 19hl. The Patholoey of
Riboflavin Deficiency in the Rat. Jr. Nutri. 22:5h5-
558-

Sherman, R.C. The Chemistry of Foods and Nutrition, 5th ed.
'New York City. 4The hacMillan Co. 1957.

 

 

-r

Snell, E.E. and Stronq. F,M. 1959. The Effect of Riboflavin
and Certain Synthetic Flavins on the Growth of Lactic
Acid Bacteria. Enzymologia 6:186-195.

‘Street, H.R. and Cowgill, G.H. 1959. Acute Riboflavin
Deficiency in the Dog. Amer. Jr. Physiol. 125:525-55h.

Street, H.R. 19h1. Studies on the Rat Crowth Assay Method
for Riboflavin. Jr. Nutri. 22:599-aoo.

Strong, F.M., Earle, Ann, and Zeman, R. l9hl. The
Distribution and Stability of Riboflavin and Pantothenic
Acid in Biolocical materials. Jr. Biol. Chem. IMO:
proc. cxxviii.

Strong, F.M. and Carpenter, L.E. l9h2. The Preparation of
Samples for the Microbiological Determination of
Riboflavin. Ind. Eng. Chem., Anal. Ed. 5h5-550.

Sullivan, Royal A. and Norris, L.C. 1959. Determining the
Riboflavin in Dried Milk Products. Ind. Eng. Chem.,
Anal. Ed. 11:555-5ho.

Supplee, G.C., Ansbacher, S. and Bender, R.C. 1955. The
Fluorometric Estimation of Lactoflavin. Jr. Biol. Chem.
110:565.

Supplee, 0.0., Bender, R.C. and Jensen, O.G. 1959.
Determininc Riboflavin. A Fluorometric and a Biological
Method. Ind. Eng. Chem., Anal. El. 11:h95.

Sure, Barnett, Dichek, fl. and Citron, M.M. 19h1. Riboflavin
as a Factor in the Economy of b'ood Utilization, I and II.
Jr. Hutri. 21:h55-h60, and.22:295-50l.

3?

Sure, Earnett, and Ford, Zenas W., Jr., 19h2. Vitamin
Interrelationships II, Thiamin and Riboflavin Inter-
relationship in Metabolism. Jr. Biol. Chem. 1L6:2h1-
250.

Van D1vne, E. 0. 19111. A Method of Dete ermi _nation in Vit
of Riboflavin in Tissues. Jr. Biol. Chem. 159:207:218.

Wanner, J. ., Axel rod, A.E., Iipton, M.A., and Elveihem,
C.A. 19 O. A R1t Assay Ietnod for the Determination
of Riboflavin. Jr. Biol. Chem. 159: 557- 5o5.

Warburc, O. and Christian, W. 1955. The Vellow Oxidizing
bnzyme. biochem. 7tschr. 256:4_9o-h99. (Nutr. Abs.
and Rev. 5:05, 1955 195L).

‘fifner, D.I., Yemmerer, A.R. and Traps, C.S. 19L2. The
Influence of Method of Preparation of Smiple on the
Microbiolo#ical Assay for Riboflavin. Jr. biol. Chem.

’9=5k7-551-

-~

Riboflavin. A Simple Fluorescent Nethod. Ind. Ens.
Chem., Anal. E4, 9:52},

We is berm, S.M. and Levin, T. 1957. The Estimation of

Whitnah, C.H., Kunnerth, B.L. and Kramer, M.“. 1937. A
Rapid Method for the Determination of Te Mtoflavin in
Yilk. Jr. Amer. Chem. Soc. 59:1155-115L

Zimmerman, H. h. and Ruwac k, E. 195R. Studies on the
Eervo1s System in Deficiency Diseases II. lesions
Produced in Docs by Diets La ackinq in the Water-Solubl
aest- Stable Vitamin a (9). Jr. Exp. Med. 59 :21- 5h.

{\J

APPENDIX

 

 

 

 

 

o o o o o o oH 9H .9 H seeeHoo
1. mac. + 0 mac. m m.a0H pH .9 N ease seamen
893. m.
m N a wee. nee. es. men. He e.mOH oeH gene: a session m.
u
a O
o ewe. o no. 0 n.H m.Hn mH .9 M aHHoe u.
a: -1 -1 no. moo. w w.na nH .9 H aoeeam
-1 one. nae. an. eo. OH n.noH ow eoqnm m eaosm
o eeH.o “so. en.n moH. an H.ns av H see
mH eo. nee. nn. awe. m o.mH on .o a noopteoe
o o moo. nu. Hmo. m.m n.em OOH .o e\n coax
0 see. 0 no. o n.H o.Hn mH .a M aHHee
o o o no. woo. w n.na mH .9 H pepeam
u- eHo. sHo. mH.n No.0 m m.wm on moamw.H eeeam "a
J
9
11 11 II II II II mm ma .9 H hamsm m.
"w
H meo.o nHo.o OH.D mno.o on m.nOH Hm .o a an.eH m.
Emmao
-1 nHo.o nmo.o He.n moo.o ow e.He on e meets
-1 see. nHH.o eH. one. es.e ec.ms an .o m .95 eoaooov
mcfipmm
Hm moo. ano. oH. mHo.o m an as .o n\H pHaamocese
.mmfi m. .mwfi .mmfi .mms .mma .mms .wsw .
pfloq w afibmam awe :OHH anyone adwoaao mmfiaoaao anmwmn whammma poem
sensooe. 1ooem naene inosa _

 

 

 

 

 

 

 

 

 

 

 

 

H amHQ 2H wQOOL mo ZOHBHmomHOo

 

 

wearer an maco pap pcmmwpo +
agony won 0
anemone econ 1|

 

 

 

 

 

 

 

 

 

 

 

 

 

Hm OH.nH OHHH new. moan OnOH Hee. Haw v.33 338
u- an. aHO. WHO. OON OH. eaO. e a.OOH mm m eoeaooo
mwsw
n we. ens. emO. Om we. eHO. OH N.mnH owH .o a cease
Imaoom
mt
O O soO. O O H. O m O.nOH On .a H aHHme m
8
J
O O O O OOHH OO. eOO. e m.omH an .a a seepam
3 ON. 96. OS. I On. OeO. OH n .aOH Oe scone N eecnm
OH we. msO. weO. OOa Om. snO. an w.ew an .o w enema scone
s ae.H msO. HMH. we OH.H eOO. eH H.nOH ONH mnamH H cranes
1- o.o one. wH. On nH.m msH.O NH O.neH OO .esea H econ esaone
.ems .msH .nme .mme .=.H a .mme .nma .mme .nsO
cave aflomfiz cfi>wHH nae swampfl> 90pm. mahonm ssfioano mmflaoamo. pnmao. mannmmfi boom Hwofi
oeoaooae -ona 1sesa -nosa _

 

 

 

 

 

ll

A.eaOOv H aaHO a

soooa an ZOHeHnOszu

 

mmomap CH mace 95L pctnmam +
sosx 902 o
pconmhm one: It

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

111.1.11.- - 511- .11 1
WW WHH HO.H OE. $3. OWOH Wee. enema eOnHW a a See
1 .l 111-- 1.1% 4,
. O -1 nHO. WWO. On Hm. WOO. OW O.HO OW m magma.
meo N
O .1 .OOO. WHO. On O nWO. n O.eaH OW mexn ossmoHooa”
. 1 . S

l O O sWO. O O nO. O n.H W.Hn WH _ .9 a aHHoe” .m

_ _ M d

. 8

_ O m O O O Onn no. . WOO. W W.nm nH .9 H noppsmu a

1- l W. L WWO. . WWO. 11 OW. eO. OH n.WOH Oe emaHe W L emote

, n .
s . We.H m WaO. . mH. We H.H eOO. sH H.nOH OWH osan H , onepoa

_ _ _mxampm mswcacoms
OH 1. . mO. WWO. OWW W. OWO. OH H.nH OO o eossoo
:1 W.e WW. * OWH. Om nH.n oaH. WH W.neH WW W mate H comm esaosom
1. I I LYI'II'IJ ~ _ ll 'J'UIIII l“!
O O _ O O _ O . O O O OH W V .p m sHpeHoc

_ fl . mean

i 1- 1- . WmO. + + O WmO. a W.WOH sH .e W . 2e are

i .

H O mmO. 11_ OWH. WsO.~ OWs on.H mOH. OW H.ns as H . was”

_ +£1-11! a . 1.11.111 'l IIIT 1
.ess ..oas m .mms .nma_.O.H W .mWe .ame nee“ . .asO w ..
pace :Homwz cabaam :HE an¢9H> coaH mahona esfloata mmfinoaun_ pnmflmu whammmH . poem .

ownaooma Ionfim uaHn Imona _ ” ~

 

 

 

 

H.92OOV HH emHo 2H WOooa aO zeHeHnOasOO

 

 

 

 

w—u—r-_—_

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

a 11 + + WWW WW.O WWO. WH W.WW WWH asse.a W WWWWWWsOL
.o .m. mane Hermon“;
O O WHO. WWOL OWW OW. WWO. WH W.OWH WW W eoessonWL n.
L n
O O WWO. O O O WO. _ W.H W.HW WH .W W WHHWOL m.
L .
L _
O O O O OWW WO. WOO. W W.WW OH .9 H seepsm L L
_ _
11 WW. WWO. WWO. 11 an. eO. OH W.WOH OW WWOHHW W eeosm
5.96% _
W.H O WOO. WOO. W WH. HO. L W.WH W.W OW LHWWW H .WaoHoO_
% _ . mOHSHL
W.eH WW. HWO. WWO WWW WW. WOO. W.W WH WW .o W opesoe_
O WW.W O O L O We . WWO. W OWH OOH .O W\W WeoonWioa _
L 1L . -h
. _ L
O O WWO. O L O WO. O L W.H W.HW L OH .9 W WHHWW L L
. L . L
11 11 L 11 11 11 11 11 11 L WH .W H semen _ m
. L L
H O L WWO. WHO. OeW OH. WWO. OW WOH HW L.o w sn.aHL
9.11.95 No L.
. L e L
O O O O L OWW WO. WOO. W W.WW WH .W H L topper u WM _
. B
L L S
11 WH. vHO. WHO. 11 WH. W0.0 W WW.W. OW ooWHW H L Oeeam e .+ ‘
L
. L
11 O WWO. WWO. 11 WW.H WWO. HH W.HOH L WW peso . emcee L .
1WWn H Ooeeosnm L L
- L L _
He 11 L OHO. WWO. HW HW. WHO. OH We _ WWH .o W ooHsW L L
panhmwomauL
L L I L
o ..-1.-.-_ n . R... r - 4‘ PM 1 w a
mo? .mb? ome ombfi .DoH 4 .mb5 .mbfi oWMé .mEU a
puma-W- nHoWHz “SSW-C HEEL cfiampfiw CPHH- anyone Ego-mt amino-WW0 Ohm-moan.” ohHm._WmW.,L- woo-m Hume.- L
oHoaooWW 1onem. 1WWWW. 1eoeW- . . L L L . L
w. L -11111, 11+

 

H4 HnHQ 2H

 

ngOh LO ZOHHHMOmwOU

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

O O O O O O O O1 O.W W .p m OHOWHWO
11 11 WOO.O O + O WOO.O OO.W O.WOH WH .W W OOOO.WWOOWO
O O OOO.O WOO.O WO.OWH WOO.O WWOO.O WO.OW O.WOH OO W OWWW opcO
menus W OWOOWO mm
W OH.O + O.O 11 WW.O OHO.O HH O.WW OOH SHAW W O.W-mm m.
O O WOO.O O O OO.O O O.H W.HO WH .W O WHHOO
O O O O OOO OO.O WO.O O.W O.OW OH .W H WWOOOW
11 WO. WWO.O WWO.O 11 WO.O OOO.O O.OH W.OOH OW mmqnm W OGOWW
W.O WW.O ‘WHO.O WO.O WWW WH.O WOO.O H.W W.W OW .Ome O\H WOHOOW OOWWWO
11 WO.O WOH.O WWO.O OWW WO.H OOH.O O.WW O.OW WW H WWW
11 11 WOO.O WOO.O 11 OW.O OOHO. WO.O HO W WOHHW H noouW
O O WOO. O O WO.O O. O.H W.HO WH .W m WHHoO
O O O O OOO WO.O WOO. W O.OW OH .W H WOOOOWL
H
J
11 OH.O OHO.O OHO.O 11 WH.O WO. O O.WO OW WOHHW H OWOOWM %
L I.
IV
11 11 11 11 11 11 11 11 WO OH .W H OWWOO O“
n..-
H O WWO.O WHO.O OOW OH.O WOO. O.OO OOH HO .0 M WO.WH EWOWO
O WWO.W O O O HW.H OOH. L O.OH WOH OO .O O\H WWOOOOWWO
L OOHSH.
OO 11 OHO.O WOH.O WW WW.O OWO. OH OW. WOH .o w WHOWWWOWWO
.WWW .OWO .WWW .WWW HfiH .O .OWW .OWW .WWO .OEO
wwog CHOWHZ nfibmam nae :fleWpH> conH mmmonn sswoawo mmflpoamo pgmwm mWSOWmE coow Hams
OOOWOOOO 1OOHWL 1WHOW 1Oosm L
41: 3.4.; ,3- ...,<._.: :1.) ,H_.,._-H-H-UD13_5)

 

 

 

C1)
0)

 

NH.MH

 

I O
n. ._ 0 Ln.
P 1 Av.

D

fi‘
03
O)

 

O

.4)
:\

B

O

..___ — ..__..._—__.__.._. '__—______ -_ _.-_ ..

CU
t-i
i)
0

5N0 0.

O

 

C:
)
ml

:0
:1")

.\J
Cu

O

 

woo.

m>a.o

0

H4808

 

a) um. o 1 ))
1"1' ‘
WHO. a

HH

C‘J

.-\~
vV

 

~r

LO

0)

t0

__,_ . ._.... -......._._—- .-

 

0

Cd

’ \
‘f'
(.1)

C

r1512

 

IIIIMkE11

m _C PM

W (.511

.DHHW

m h. E

WEE

mmflboow
hmtmfiu

 

. .-a1L
H .rwkxpLCOm

pflbhn

haam?

l

I' \
t.”
$5»;

WWOO

rU
m
C)
£4
£3!

Shoo
wmgcwg
0pwwc$

WOOL
UC§OHU

L
L
L

(D 'r"
g
L
FL.

 

DHo<
0meoom<

 

 

_C H O m H a.

 

OOOWHW
1OOHO

 

 

 

A.QCOOL HHH NY

d CHE
I m p 1w ..._.1

 

 

mSLOfiQ
1 mew-E

 

ESWO
1HWO

 

 

L

\
P“
1

 

 

 

—

1H flfiqom n:

TQHHwflQh-QU

 

JGUUIG

v
',:_-.

 

 

UHLI

.-

 

., w
‘ 1. n:
o ,
'1. .§
"I :1.

 

CllHlGAlN STATE UNIVEILRSITY

111111 111 ”58:11

 

m "‘"o- .041— : a—s are. “'WAH -v~.x