Hemolytic complement activity in the thiamine deficient guinea pig

Item Type text; Thesis-Reproduction (electronic)

Authors Crisman, Jon Eliot, 1939-

Publisher The University of Arizona.

Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.

Download date 01/10/2021 07:34:09

Link to Item http://hdl.handle.net/10150/318633 HEMOLYTIC COMPLEMENT ACTIVITY IN THE

THIAMINE DEFICIENT GUINEA PIG

by Jon Eliot Crisman

A Thesis Submitted to the Faculty of the

DEPARTMENT OF MICROBIOLOGY AND MEDICAL TECHNOLOGY

In Partial Fulfillment of the Requirements For the Degree of

MASTER OF SCIENCE

In the Graduate College

THE UNIVERSITY OF ARIZONA

19 6 7 STATEMENT BY AUTHOR

This thesis has been submitted in partial fulfill­ ment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Libraryo

Brief quotations from this thesis are allowable without special permission, provided that accurate acknowl­ edgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the inter­ ests of scholarshipo In all other instances, however, permission must be obtained from the author«

SIGNED:

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

,A / y WAYBURN/S/ JETER B'ate4- ~ Professor of ^Microbiology and Medical Technology ACKNOWLEDGMENTS

The writer wishes to express his sincere apprecia­ tion to Dr o Wayburn So Jeter for his guidance and assistance throughout this study»

iii TABLE OF CONTENTS

Page

LIST OF ILLUSTRATIONS ...... v

LIST OF T A B L E S ...... vi

ABSTRACT ...... vii

INTRODUCTION ...... 1

MATERIALS AND METHODS ...... 7

Animals ...... 7 Test D i e t s ...... 7 Hematology ...... 9 Blood Glucose ...... 9 Serum Protein ...... 10 Hemolysin Titration ...... 10 Hemolytic Complement Titration . 11

RESULTS ...... 13

DISCUSSION ...... 31

SUMMARY ...... 34

LIST OF REFERENCES ...... 35

iv LIST OF ILLUSTRATIONS

Figure Page

1. Weight changes seen in two weanling guinea pigs during evaluation of test diets . l4

2 o Weight changes seen in the 4 thiamine deficient animals in Group I ...... 17

3rJ>~ Wehght changes ,seen in the 3 inanition control animals in Group I . 17

4 o Weight changes seen in the 3 thiamine deficient animals in Group II * l8

5 o Weight changes seen in the 3 inanition control animals in Group II » 18

6 o Weight changes seen in the 6 thiamine deficient animals in Group III ...... 19

7 o Weight changes seen in the 5 inanition control animals in Group I I I ...... 19

8. Weight changes seen in the 2 normal control animals in Group loo,...... 21

9 o Weight changes seen in the 2 normal control animals in Group I I ...... 21

10, Weight changes seen in the 3 normal control animals in Group I I I ...... 22

v LIST OF TABLES

Table Page

1• Composition of the Reid-Briggs test diet * . . 8

2 o Hemolytic complement activity in serum of Group I animals fed various d i e t s ...... 23

3 o Hemolytic complement activity in serum of Group II animals fed various diets ...... 24

4 o Hemolytic complement activity in serum of Group III animals fed various diets * . „ • 25

5« Summary of serum protein values for animals on various diets ...... , • . » « • 26

6 o Summary of serum glucose values for animals on various diets ...... 27

7 - Summary of total leukocyte counts for animals on various diets ...... 29

8• Summary of hemoglobin values for animals on various diets . , 30

vi ABSTRACT

Male guinea pigs of the Amana strain grew normally when fed the complete Reid-Briggs diet from weanling age.

This same diet lacking in thiamine HCl produced a well- defined thiamine deficient state in the 400-500 g animal with signs of anorexia appearing after 10-15 days and a fatal termination in 30-50 days. No significant alteration in hemolytic complement activity9 serum protein, serum glucose, or total leukocyte count could be demonstrated in these thiamine deficient animals as compared to inanition and normal control animals. An increased hemoglobin level representing hemoconcentration was found in the deficient animals and in some of the inanition controls. INTRODUCTION

Zilva (1) was one of the first investigators to recognize the complex nature of resistance to infectious diseaseo He proposed the study of effects of such variables as diet on individual body defense mechanisms rather than on general resistance to infection. Numerous investigators have since studied the effects of both general undernutrition and specific nutritional defi­ ciencies on such mechanisms yas antibody production, cellular blood composition, cellular migration in inflamma­ tion, phagocytosis, and complement activity.

A recent review by Axelrod and Pruzansky (2) summarized the results of investigations of antibody synthesis in deficiency states. In vitamin A deficiencies, impaired antibody synthesis has been reported by Green (3 ),

Ludovici and Axelrod (4), and Panda and Combs ($), whereas

Natvia (6), Zilva (l), Feller et al. (?)? and Underdahl and

Young (8) found no such effect. Natvia (6) and Zilva (1) found no reduction of antibody production in the scorbutic guinea pig. Madison and Manwaring (9 ) reported that injec­ tion of large doses of vitamin C into rabbits caused a twenty-fold increase in antibody against horse serum. Most investigators (3, 4, 6) agree on the failure of vitamin D to alter antibody production but Pruzansky and Axelrod (10) found impaired diphtheria antitoxin production in the vitamin D deficient rat• Wertman and coworkers (11, 12,

13, l4), Axelrod et a l » (1 3), Carter and Axelrod (l6),

Ludovici and Axelrod (4), and Axelrod and Pruzansky (17) have demonstrated a requirement for each of the vitamins of the B-complex group«

A second area of investigation has dealt with phagocytosis and the related subjects of cellular blood and exudate composition. Green (3 ) found a marked leukocytosis in the rabbit in vitamin A deficiency but not in vitamin D deficiency. Wertman and coworkers (l8, 19,

20, 21, 22, 2 3 ) found the total cell and differential counts in peripheral blood and in peritoneal exudates to be variously altered in rats made deficient in thiamine, riboflavin, pyridoxine, folic acid, niacin-tryptophan, and vitamin , with less marked but similar effects in the inanition controls for all deficiencies except pyridoxine.

In these studies, the results of bone marrow examinations reflected the changes found in the peripheral blood.

Reduced phagocytic activity has been reported with rats deficient in riboflavin, pyridoxine, pantothenic acid, choline, and thiamine (23, 24, 2$). Natvia (6) and Feller et al. (?) have reported no change in phagocytic activity with vitamin C deficiencies in guinea pigs and human beings respectively, whereas Cottingham and Mills (25) found reduced in vitro phagocytosis of Micrococcus candidus in the scorbutic guinea pig.

In the study of hemolytic complement activity in relation to nutrition 9 March (2 6 ) reported the partial or complete loss of activity in guinea pigs deprived of vitamin C for only seven days and Morgan (2?) noted that guinea pigs deprived of grass or other fresh food had a lowered titer which could be raised by feeding mangolds or cabbage as sources of vitamin C . In the study of human subjects^ Chu and Chow (2 8 ) found that lowered serum vitamin C levels coexisted with reduced complement activity and that the administration of ascorbic acid resulted in a rise in both vitamin C and complement levels in the blood„

Ecker et al. (29) and Ecker and Pillemer (30) reported a direct correlation between serum vitamin C concentrations and complement activity in both guinea pigs and human beings. These authors also reported that in vitro addition of ascorbic acid to serum increased both the complement titer and the stability of the complement on storage.

Ecker et al. (31) found that serum complement inactivated by chemical agents or by aging for up to eight days could be reactivated by the addition of vitamin C .

In contrast, Agnew, Spink, and Mickelsen (3 2 , 33) found complement activity in human beings and guinea pigs

j unchanged by either a vitamin C deficiency or excess in vivo or in vitro vitamin C levels. Crandon, Lund, and Dill (34) and Feller et al. (?) also have found no loss of complement activity in vitamin C deficient human beings «, and

Natvia (6 ), Kodicek and Traub (35) 9 and Rangam«, Chandra, and Chowdhary (3 6 ) all reported no significant change in the complement levels of scorbutic guinea pigs 0

Vitamin D deficiency is reported to have no effect on complement activity in the rabbit (3 ) or rat (6). In vitamin A deficiencies, complement activity is reported to be unchanged in human beings (7)9 guinea pigs (3 6 ), rabbits

(3)9 and rats (6, 37)« ' However, Osborne (3 8 ) found a tendency toward reduced activity in rats deficient in both vitamin A and ftsome other factortl which was found in cod liver oil but which was thought not to be vitamin D •

Rose and Kolmer (39) reported no change in comple­ ment activity with E-complex avitaminosis resulting from feeding dogs a diet containing 15% of the minimal B-complex requirements for a period of three months. Natvia (6), likewise, found no change in complement levels in B-complex deficient rats. More recently, Wertman and coworkers (l8,

19 ? 20, 21, 40) have reported the complete loss of activity in niacin-tryptophan deficiency, partial loss of activity in thiamine, pyridoxine, riboflavin, and folic acid defi­ ciencies, and no change in activity with vitamin B^^ deficiency in the rat. In these experiments, reduced activity was also noted in the respective inanition controls but to a lesser extent. 5

In similar experiments, Axelrod and Pruzansky (17,

4l) reported that biotin and pteroylglutamic acid defi­

ciencies in rats had no effect on complement activity and

attributed the lowered activity observed in pyridoxine and riboflavin deficient rats to inanition rather than to the

specific deficiency. These authors also studied the regeneration of complement in rats depleted by the daily

intraperitoneal injection of pneumococcal polysaccharide

followed by intravenous injection of homologous antiserum.

Complement activity returned to the predepletion level at

the same rate in both deficient and control animals.

In the study of complement in protein deficiencies,

Eangan et al. (3 6 ) reported a gradual but significant

reduction in activity with methionine and cystine defi­

ciencies of 40 days duration in the guinea pig, and Kenney

et al. (42) found a slow decrease in activity in the rat

over a 10 week period with various protein-depletion diets.

Weimer et al. (43) found that in semistarvation the

complement activity in rats decreased with a 12% weight

loss, increased with a 2 6 -33% weight loss, and returned to

normal with repletion. In a later study, Weimer et al.

X 48) found increased activity with weight losses of 12% and

26% caused by inanition and decreased activity with a

weight loss of 42% due to protein depletion.

The present sta£;e of knowledge concerning the

relationships of nutrition to body defense mechanisms has become quite confused« This is primarily the result of the failure of many investigators to use standard test animals 9 diets 9 dietary regimens , etc» The data concerning comple­ ment activity in deficiency states are especially difficult to interpret. The guinea pig is the animal of choice for such studies because of its high hemolytic complement activityo However, due to difficulties encountered in adapting this animal to experimental diets, most investi­ gators have employed other experimental animals. The purpose of this study was to determine the effect of a thiamine deficiency on complement activity in the guinea MATERIALS AND METHODS

Animals

The animals used in these experiments were male

guinea pigs of the Amana strain« All animals were raised from weanling age in individual metal cages with screened bottoms and remained in these cages throughout the study.

Fresh tap water containing 5 mg ascorbic acid/100 ml was provided ad libitum to all animals« Blood samples of

4-g ml were obtained by means of cardiac puncture under

eiher anesthetic•

Test Diets

Reid-Briggs Guinea Pig Test Diet (General Biochem*

Corp.) was employed as the complete ration. This diet without added thiamine HC1 was employed as the thiamine

deficient ration. The ingredients contained in the

complete test diet are given in Table 1.

From weanling age, all animals were fed the

complete diet ad libitum until each had attained a size

suitable for use. The guinea pigs were then divided into

thiamine deficient, inanition control, and normal control

groups. The normal controls received the complete diet

ad libitum throughout the experimental period. The

thiamine deficient animals were offered the thiamine Table 1. Composition of the Reid-Briggs test diet^

Ingredients lbs/100 lbs Ingredients g/100 lbs

Casein, Plain, not vitamin-free 30.00 Thiamine HC1 0 . 7264 Corn Starch 20.00 Riboflavin 0.7264 Sucrose 10.30 Pyridoxine HC1 0.7264 Glucose 7.80 Calcium Pantothenate 1.8160 Non-nutritive Fibre _( cellulose ) 15.00 . Niacin 9.0800 Corn Oil 7.30 Biotin 0.0272 Salt Mixture (Fox^Briggs) 6 .00 Folic Acid 0.4540 Potassium Acetate 2.50 Vitamin 6^2 1 .8l60 Magnesium Oxide 0.50 Vitamin A Acetate 0.2724 Choline Chloride 0 . 20 Alpha-tocopherol i-Xnositol 0.20 Acetate 0.9080 Manadione 0.0908 Calciferol O.OOI816 Ascorbic Acid 90.8000

aAnalysis given by supplier (General Biochem. Corp.). deficient test diet ad libitum, and for each deficient

animal a weight paired inanition control was fed the

complete diet in amounts sufficient to maintain a weight

gain or loss similar to that of the deficient animal« No

other food was offered«

Hematology

Total leukocyte counts were performed using the

Model B Coulter Counter and employing a modification of the method of Richer and Breakell (45) in which 0,02 ml of whole blood was added to 10 ml filtered saline containing

0,04% formalin. Prior to counting, 0,05 ml of a 2 g/100 ml

solution of saponin in saline was added and a period of

15-60 minutes was allowed for hemolysis to take place. The

samples were counted using threshold settings of 20-low and

100-high, current 0,707, and amplification of 1, Hemo­

globin concentration was measured by the cyanmethemoglobin method of Crosby, Munn, and Furth (46) using whole blood.

Blood Glucose

A protein-free filtrate was prepared from serum

using the method of Boutwell (47) in which 0,2 ml of serum

is added to 1,6 ml of N/12 H^SO^ followed by the addition

of 0,2 ml of 10% w/v sodium tungstate. Glucose was

determined by a modification of the anthrone method of

Umbreit, Burris, and Stauffer (48) using 1,0 ml of a 1:10

dilution of the protein-free filtrate in 2,0 ml of anthrone 10

reagent• The mixture was boiled for 3 minutes 9 cooled, and

the color density measured at 6 50 m\l in a Coleman Junior

Spectrophotometer «

Serum Protein

Total serum protein was measured using a modifica­

tion of the biuret method of Gornall, Bardawill, and

David (49). To 0.1 ml of serum was added 1.9 ml of saline

and 8.0 ml of biuret reagent. The mixture was allowed to

stand for 30 minutes and the color density was measured at

540 m[i in a Coleman Junior Spectrophotometer.

Hemolysin Titration

Sheep erythrocytes were obtained from ewes by

bleeding aseptically from the jugular vein into an equal volume of sterile Alsever1s solution. The suspension was

aged for 4 days at 4 C and was used within a 30 day period.

Prior to use, the cells were washed three times in 0.15

M NaCl and then resuspended to a concentration which, when

diluted with four parts distilled water, gave an optical

density of 0.250 at 520 m[i in a Coleman Junior Spectro­

photometer.

Rabbit anti-sheep RBC hemolysin was prepared

according to the method of Darter (50) and stored at -20 C

in an equal volume of neutral glycerin until used. The

hemolysin was titrated using a modification of the method

of McKee and Jeter (51)« The highest dilution of hemolysin 11

giving complete lysis of sheep RBC1s following incubation

at 37 C for 30 minutes in the presence of a constant excess

of guinea pig complement was considered one hemolytic unit o

A protocol for such a titration follows:

Controls Tube # 1 2 3 6 9 Pos Neg

Hemolysin Dil.

(0.2 ml) 1:2000 1:3000 1:4000 1:7000 1:10,000 1:1000 — — — —

Comple­ ment 0.2 ml 0.2 ml 0.2 ml 0.2 ml 0.2 ml ------0 o 2 ml

Mg- saline 0.4 ml 0.4 ml 0.4 ml 0.4 ml 0.4 ml 0.6 ml 0 o 6 ml

Sheep RBC 0.2 ml 0.2 ml 0.2 ml 0.2 ml 0.2 ml 0.2 ml 0 o2 ml

al.25 millimolar MgSO^ in 0.15 M N a C l .

Hemolytic Complement Titration

Blood for complement titer was allowed to clot and

the serum separated at refrigerator temperature„ Clear

serum thus obtained was stored in an ice bath and the

titration was completed within four hours of sample

collectiono Hemolytic complement activity was measured

according to a modification of the method of McKee and

Jeter (51)° Varying amounts of diluted serum were added to

sensitized sheep RBC1s in a constant total volume of

1 * 0 m l » Following incubation at 37 C for 30 minutes % the 12

tubes were cooled by immersion in an ice bath and then

centrifuged for 5 minutes at 350 X g at 4 C in an Inter­ national Refrigerated centrifuge. The optical density of the supernatent fluid was measured at 520 m[l in a Coleman

Junior Spectrophotometer and the 50% hemolytic endpoint j determined. The sensitized erythrocytes used were prepared by mixing a standardized sheep RBC suspension with an equal volume of hemolysin containing two hemolytic units per

0.2 ml. The mixture was allowed to stand at room tempera­ ture for 15 minutes prior to use. A protocol for a typical titration follows:

Tube # 1 2 3 ^ 6 8 10

Diluted Serum, ml 0.10 0.09 0.08 0.07 0.05 0.03 ----

Saline, ml 0.10 0.11 0.12 0.13 0.15 0.1? 0.20

Mg-saline, ml 0.40 0.40 0.40 0.40 0.40 0.40 0.40

Sen. SRBC, ml 0.40 0.40 0.40 0.40 0.40 0.40 O.40 RESULTS

Preliminary experiments were devoted to the evaluation of the complete and deficient test diets.

Eighteen adult, male guinea pigs, aged 6-7 weeks and weighing 530-640 g, were used in the first trial feedings.

Ten of these animals were offered the complete test diet and eight received the laboratory ration normally fed

(Wayne Guinea Pig Chow). Although their appearance remained essentially normal, the animals on the Reid-Briggs diet lost 1.6 g/day while the guinea pigs receiving the lab chow gained 4.1 g/day during the three week period.

Failure of the animals on the complete test diet to gain at a normal rate appeared to be due to refusal to eat rather than to any nutritional inadequacy of the test diet.

Accordingly, the next evaluation was performed using two weanling animals which were l4 days old and weighed 272 and 302 g respectively. Figure 1 shows the weight changes observed with these animals over a 13 week period in which the complete diet in both pelleted and powdered form and the thiamine deficient test diet in powdered form were evaluated. These two guinea pigs were started on the pelleted complete diet and, for the first week, were hand fed several pellets each day to promote an interest in the new diet. As can be seen, these weanlings

13 Figure 1. Weight changes seen in two weanling guinea pigs during evaluation of evaluation during pigs guinea weanling two in seen changes Weight 1. Figure

BODY WEIGHT (G) et diets. test 250 500- 400- 550- 450- 300- 350- 20 30 IE (DAYS)TIME 40 haieDfcet it Fed Diet Deficient Thiamine — Fed Diet Reid-Briggs Complete — ® Animal #2 Animal ® #1 Animal ° Thiamine HC1 Injected IP Injected HC1 Thiamine 6050

70 80 90

15 responded well to the new diet, gaining 4,1 g/day. After three weeks of pellets, they were offered the complete diet as a powder and again fared well with an average weight gain of 6,2 g/day and a normal physical appearance.

After six weeks of the complete diets, the two guinea pigs weighed 473 and 533 g« They were then placed on powdered, deficient test diet and observed for an additional five weeks. Both animals had begun losing weight within 10 days of this change in diet. By the 20^ day, anorexia was marked and at 30 days the animals had lost 115 and 177 g respectively. Both were severely emaciated, tended to by hyperreactive to noise, had roughened coats, and the skin of their^feet appeared abraded and tender. Both animals had difficulty main­ taining their balance and by the 32nd day appeared near death.

Thiamine HC1 was then injected intraperitoneally in amounts of 1 mg/day for five days. The two animals responded quickly to the thiamine injections with the return of their appetite followed by pronounced weight gain. They were then offered the complete diet again and at the end of the trial period both had regained most of the weight lost and had returned to a near normal appear­ ance.

Studies of hemolytic complement activity were carried out using three groups of animals. For each test l6 group, weanling animals were placed on the complete diet for a period of adaptation and those which gained weight at an acceptable rate were then included in the study* A total of 4? weanlings weighing an average of 2^9 g were started on the diet. Eleven of these gained an average of only 1.5 g/day and were subsequently excluded. The weanlings that successfully adapted to the base diet gained an average of 5-9 g/day.

Each experiment was begun when the weanlings reached a weight of approximately 400 g on the complete diet. As shown in Figure 2, the deficient animals in Group

X continued to gain weight for the first 10-15 days on the deficient diet and then three of these animals lost weight at the rate of 6.1 g/day throughout the remainder of the test period. The fourth animal responded to the deficient diet only by a reduced rate of gain for the remainder of the test period. In order to cause a weight loss in the inanition controls (Figure 3) comparable to that of the deficient animals, rations were reduced to 3“5 g/day from the 10ti* to 40ib days and thereafter withheld completely.

The deficient animals in Groups II and H I (Figures

4 and 6) responded to the deficient diet somewhat differ­ ently than did the Group I animals. These animals also continued to gain for 10-15 days and then lost weight at the rate of 5-4 g/day (Group II) and 9-3 g/day (Group III).

The two experiments were terminated after 29 and 31 days Figure 3. Weight changes seen in the 3 inanition control 3 inanition the in seen changes Weight 3.Figure

BODY WEIGHT CG) • BODY WEIGHT (G) 300 egt hne se n h 4 haie deficient 4 thiamine the in seen changes Weight nml i ru . I Group in animals nml i ru I.Group in animals 400- 400- 600- 600- 500- 400- 500-. o-— cr 10 10 IE (DAYS) TIME 20 20 IE (DAYS) TIME 30 30 40 0 4

17

18

<£> 600- h- X o

1x1 500-

>- O o 400- jzr m

300 - 10 20 30 40 TI ME (DAYS) Figure 4. Weight changes seen in the 3 thiamine deficient animals in Group II.

w 600- H X o

1x1 500-

>- Q o 400-i CD

0 20 30 40 TIME ( DAYS)

Figure 5* Weight changes seen in the 3 inanition control animals in Group II. iue 7• control Figure 5 inanition the .in seen changes Weight BODY WEIGHT (G) g BODY .WEIGHT CGJ) ue . egt hne se i te timn deficient thiamine 6 the in seen changes Weight 6.gure 500- 400- 300- 400- 300- 500- animals in Group III. Group in animals animals in Group III.Group in animals 10 10 20 20 30 30 TIME TIME C DAYS) •y/ (DAYS: 10 10 20 20 0 3 30

19

20 respectively as compared with 48 days for the Group I experimento In order to match this weight loss 9 it was necessary to completely withhold rations from the inanition controls after the l8tb day for Group II (Figure 5 ) and 22nd day for Group III (Figure 7)• The normal controls in these three groups (Figures 8-10) gained at the rates of 5*3 g/day (Group I), 5 « 5 g/day (Group II), and 8*0 g/day (Group

III) o

The first physical signs of the thiamine defi­ ciency other than weight loss were marked anorexia and roughened coats observed after 18-22 days with all three groups o The animals then became increasingly emaciated, excitable, and tended to lose their balance. The inanition controls also became emaciated and had roughened coats but showed none of the other signs seen in the deficient animals. Ten of the deficient guinea pigs were so weakened that they failed to recover from the anesthetic after the final bleedings and two of the remaining deficient animals died during the following 24 hours. Only three of the inanition controls failed to survive the final bleeding.

Tables 2-4 show the hemolytic complement activity measured throughout the study. As can be seen, there is considerable variation between animals, between groups, and between samples obtained from the same animal on different dates. Variation is also seen in the serum protein (Table

5 ) and serum glucose (Table 6 ) values. As gross inspection 21

O ^ 800- H X (£> w 700-

> Q O 600- m

500. 10 20 30 40 TIME (DAYS)

Figure 8. Weight changes seen in the 2 normal control animals in Group I .

O 600- h- x o LU 500-

g 400- m 300 10 20 30 4 0

TIME tDAYS)

Figure 9• Weight changes seen in the 2 normal control animals in Group II. iue 0 Wih changes Weight seenin the 3 normal control Figure 10.

BODY WE! GHT (G) animals inGroup III. 400 600- 700- 500- T 20 1 M (DAYS) ME 0 4 0 3

22 Table 2, Hemolytic complement activity in serum of Group I animals fed various diets«

Subgroup

Thiamine Deficient Inanition Control Normal Control

Animal # Sample 44 46 40 42 4l 48 53 43 51

Units/ml S erum C 'H50 Day 0 888 1395 . 1332 1380 1380 1092 1350 1092 1332

Day 5 1090 1332 1445 1480 1504 1125 1335 1320 1190

Day 11 1040 1310 1370 1460 1540 1300 1660 1400 1180

Day 18 1105 1130 1200 1380 1360 1285 1320 1130 1105

Day 3 4 1060 1060 1340 1055 1250 1265 1240 ll80 1210

Day 4l 1060 1130 1100 984 1130 1190 1060 10f>0 1080

Day 46 1210 1290 Died 840 1530 Died 1315 1518 Died

Day 50 1100 Died -- Died 1080 -- 700 1285 --

ro V) Table .3* Hemolytic complement activity in serum of Group XI animals fed various diets.

Subgroup

Thiamine Deficient Inanition Control Normal Control

Animal # S ample 76 78 80 79 81 85 73 83

C'Hj_q Units/ml Serum

Day 0 984 864 857 882 992 960 984 857

Day 7 833 839 861 926 1010 1040 985 1010

Day 12 970 865 1175 1120 885 1035 1000 1080

Day 23 875 1030 900 860 io4o 1110 1040 1075

Day 29 Died 825 io4o 1175 Died 940 Died 1125 Table 4. Hemolytic complement activity in serum of Group XXI animals fed various diets o .

Subgroup

Thiamine Deficient Inanition Control Normal Control

Animal # Sample 4 6 7 16 17 19 10 10 12 15 18 2 3 13

C »H,-0 Units/ml Serum

Day 0 985 1300 1240 1070 860 1060 1420 1130 1090 1220 1070 1270 1130 1260

Day 7 1060 1290 1290 1060 -- -- 1060 860 1160 940 -- 1200 1240 1060 O O H Day 31 1370 1590 1380 915 94o VI 1280 955 i44o 1160 810 1370 1270 1280 26

Table 5- Summary of serum protein values for animals on various diets.

Thiamine Inanition Normal Deficient Control Control

mg/ml Serum

Avg 63.5 6 3 .I 60.2 Group I: Range (5 3 .5 -7 6 .0 ) (54.5-92.0) (5 2 .0 -7 2 .0 )

Avg 62.1 61.0 60.1 Group I I : Range (5 4 .0 -7 1 .0 ) (54.0-77.0) (48.0-63.0)

Avg 58.8 75.5 60.7 Group III Range (4 7 .5 -6 6 .0 ) (46.0 -7 1 .0 ) (56.5-67.5) 27

Table 6„ Summary of serum glucose values for animals on various diets

Thiamine Inanition Normal Deficient Control Control

mg/100 ml Serum

Avg 186 183 195 Group I : Range (107-327) ( 90-437) (142-315)

Avg 159 l80 183 Group X X : Range (1 0 0 -3 8 0 ) (127-317) (115-290)

Avg 138 129 172 Group I I I : Range (107-2X0) ( 80-167) (140-210) failed to show any conclusive trends, these data were transformed to L o g ^ values and subjected to statistical analysis by means of analysis of variance with each test group being evaluated separately- Within each group the data for the deficient, inanition control, and normal control animals were pooled separately by date of bleeding and each of these pools was considered a single , treatment«

At the 95% confidence level, there was no significant change in complement activity, serum protein concentration, or serum glucose concentration in the deficient, inanition control, or normal control animals throughout the study -

The results of the hematological examinations are given in Tables 7 .and 8 - The total leukocyte counts remained essentially normal in all animals throughout the study - In each of the three groups, the hemoglobin concentration in the deficient animals increased from an average of 13-1 g/lOO ml in the initial blood samples to an average of 15-2 g/100 ml in the final samples- A similar increase in hemoglobin concentration is seen in the inanition control animals of Group II, but in all other

animals the hemoglobin concentration remained essentially unchanged - 29 Table 7« Summary of total leukocyte counts for animals on various diets.

Thiamine Inanition Normal Deficient Control Control

WBC/mm^ Whole Blood

Avg 4833 5X09 44X5 Group I : Range (3X27-7625) (3505-6948) (3317-5041)

Avg 4o4o 4360 5615 Group X I : Range (2402-6344) (3X27-5749) (4684-6394)

Avg 3670 4942 4289 Group X X I : Range (2394-4522) (3717-5389) (3281-5569)

i 30

Table 8, Summary of hemoglobin values for animals on various diets*

Thiamine Inanition Normal Deficient Control Control

gm/100 ml Whole Blood ( avg)

Prebleed 12*9 14.3 13-1 Group I: Final 15 • 5 13.0 13.6

Prebleed 12*7 12.4 12.8 Group II: F inal 15.3 15.6 13.5

Prebleed 13-6 12.7 13.6 Group X X I : F inal 14.9 12.4 13.2 DISCUSSION

In studies leading to the development of the diet used in the present study, Reid and Briggs (52) found that

2-4 day old guinea pigs gained at an average rate of

7 o1 g/day to reach an average weight of 458 g at eight weeks of age. The weight gains seen in the present study, both during the initial evaluation of the diets and with the normal controls employed in the deficiency studies, permit the reasonable assumption that the Reid-Briggs diet is nutritionally adequate for the guinea pig.

Using the same diet, Reid (53) described a well- defined thiamine deficiency state in guinea pigs of the

Hartley strain. Again starting with 2-4 day old animals, an average survival time of 24 days on the deficient diet was found. Physical signs of the deficiency were anorexia, emaciation, tremor, and spasm. These findings are essen­ tially the same as we found and they support the conclusion that an acute thiamine deficiency was produced.

Several explanations are suggested for the failure of the thiamine deficiency and the accompanying starvation to alter complement activity. Thiamine may well not be required for the maintenance of normal complement levels in the guinea pig. Conversely, thiamine may be required, but the thiamine levels remaining even in a severe

31 32 deficiency may be sufficient to sustain normal complement / activity. Residual thiamine concentrations of 10-25% of normal have been reported in various tissues of the deficient rat (5^)• Related to the later possibility is the suggestion that certain key body components are selectively maintained during periods of nutritional stress even at the expense of other less important functions•

Such a mechanism may explain the lack of change in serum proteins as well as complement even in the terminal stages of the deficiency. ;The acute starvation state induced by the thiamine deficiency undoubtedly produced a variety of other nutritional deficiencies of differing degrees, especially in the terminal stages. That this extreme degree of nutritional stress failed to affect the comple­ ment levels of these animals is somewhat surprising.

Still another possibility may be that the rate oft turnover of the several components of complement is so slow that partial or complete inhibition of synthesis would not be reflected in serum complement activity during the relatively short duration of the deficiency. This concept would also require that there be little utilization of pre­ formed components either through immunological reaction or due to some other function during the experimental period

and would be inconsistent with current concepts of metabolism. 33 The use of guinea pigs of a different strain or age or the modification of some other test condition may pro­ duce wholly different results• In the same light9 the findings by several authors of significant alterations in complement activity in the deficient rat are not directly comparable with the results of this study and may suggest basic differences in the complement mechanisms of these two animals. SUMMARY

Weanling guinea pigs raised on the Reid-Briggs complete diet appeared normal and healthy in all respects*

Feeding the thiamine deficient Reid-Briggs diet resulted in a deficiency characterized by anorexia, emaciation, loss of balance, and eventual death of the animal *

An acute thiamine deficiency in adult, male guinea pigs of the Amana strain caused no significant alteration in hemolytic complement activity, serum protein, serum glucose, or total circulating leukocyte count *

Increased hemoglobin levels were found in the deficient animals and several of the inanition controls*

34 LIST OF REFERENCES

Zilva, S o So 1919• The influence of deficient nutrition on the production of agglutinins 9 complement«, and amboceptor» Biochem, J « 13:172c

2 o Axelrod , A.« E » and J « Pruzansky o 1955 • The role of vitamins in antibody production. Vitamins and Hormones 13:1•

3- Green, M. R» 1933• The effects of vitamins A and D on antibody production and resistance to infection. Am o J. Hygiene 17•60 »

Ludovici, Po P * and A. E * Axelrod. 1951• Circulating antibodies in vitamin-deficiency states. Pteroyl- glutamic acid, niacin-tryptophan, vitamins B^2 ^ A, and D deficiencies. Proc« Soc. Exp. Biol. Med. 7 7 :5 2 6 .

5o Panda, B. and G. F . Combs. 1 9 6 3 • Impaired antibody production in chicks fed diets low in vitamin A , pantothenic acid, or riboflavin. Proc. Soc. Exp. Biol. Med. 113:530.

6 . Natvia, H. 1942. The importance of vitamins for the resistance of the organism against bacterial infections. Nutr. Abst. and Reviews 12:198.

7- Feller, A. E., L. B . Roberts, E. P. Ralli, and T. Francis, Jr. 1942. Studies on the influence of vitamin A and vitamin C on certain immunological reactions in man. J . Clin. Investigation 21:121.

8 o Underdahl, N. R. and G. A. Young. 1956. Effect of dietary intake of fat-soluble vitamins on intensity of experimental swine influenzae virus infection in mice. Virology 2:4l5*

9 Madison, R. R. and W . H. Manwaring. 1937« Ascorbic acid stimulation of specific antibody production. Proc. Soc. Exp. Biol. Med. 37:402.

10 o Pruzansky <, J. and A. E . Axelrod. 1955 • Antibody production to diphtheria toxoid in vitamin defi­ ciency states. Proc. Soc. Exp. Biol. Med. 8 9 :3 2 3 .

35 36

11. Wertman, K . 9 F. Crisley, and J . L. Sarandria. 1952. Complement-fixing murine typhus antibodies in vitamin deficiency states. III. Riboflavin and folic acid deficiencies. P r o c . Soc. Exp. Biol. Med. 80:404.

12. Wertman9 K . and J . L . Sarandria. 1951a. Complement- fixing murine typhus antibodies in vitamin defi­ ciency states. Proc. Soc. Exp. Biol. Med. 7 6 :3 8 8 .

13. Wertman? K . and J . L . Sarandria. 1951b. Complement- fixing murine typhus antibodies in vitamin defi­ ciency states. II. Pyridoxine and nicotinic acid deficiencies. Proc. Soc. Exp. Biol. Med. 7 8 :3 3 2 «

14. Wertman, K . and J . K . Sarandria. 1952. Complement- fixing murine typhus antibodies in vitamin defi­ ciency states. IV. Bp2 deficiency. Pr o c . S o c . Exp. Biol. Med. 81:395.

15- Axelrod 9 A. E ., B . B. Carter, R. H. McCoy, and R . Geisinger. 1947° Circulating antibodies in vitamin deficiency states. I. Pyridoxine, ribo­ flavin, and pantothenic acid deficiencies. Proc. S o c . Exp. Biol. Med. 66:137* l6 . Carter, B. B. and A. E. Axelrod. 1948. Circulating antibodies in vitamin deficiency states. II. Thiamine and biotin deficiencies. Proc. Soc. Exp. Biol. Med. 6 7 •4l6.

17- Axelrod, A. E. and J . Pruzansky. 1955* The role of vitamins in antibody production. Annals N. Y. Acad. Sci. 63*202.

1 8 . Wertman, K . and M. Groh. 1959 * The effects of thiamine deficiency on some physiologic factors, phagocytosis and susceptibility to infection. J . Immunol. 8 2 :24l.

19. Wertman, K . F ., R . J . Lynn, and D . T. Bisque. 1957* The effects of vitamin deficiency on some physi­ ological factors of importance in resistance to infection. IV. Riboflavin deficiency. J. Nutrition 6 3 •311.

2 0 . Wertman, K ., R. J . Lynn, D . T. Bisque, G . W. Rohr, and M. E. Carroll. 1956. The effects of vitamin defi­ ciency on some physiological factors of importance in resistance to infection. III. Vitamin Bn 2 and folic acid deficiencies. J . Nutrition 60:473* 37

2 1 o Wertman<, K. ^ W. M . 0 1 Leary 9 and L . W Smith . 1955 « The effects of pyridoxine deficiency on some physiological factors of importance in resistance to infection. J . Nutrition 57 - 203»

2 2 o Wertman9 K. , R. Rotundo ? and R . Yea. 1953 ® Blood and bone marrow study of vitamin deficient rats. J . Nutrition 50:479*

23<> Wertman9 K . F. and P. S. Sypherd. i9 6 0 . Effects of riboflavin on phagocytosis and susceptibility to infection. J . Immunol. 8 5 •511.

24. Furness, G. and A. E. Axelrod. 1959* Effect of deficiencies of thiamine, pyridoxine, and pantothenic acid on the macrophage of the rat. J . Immunol. 83*133*

25 • Cottingham, E. and C. A. Mills. 1943* Influence of environmental temperature and vitamin-deficiency upon phagocytic functions. J . Immunol. 47*493*

26 o March, F. 1936. Ascorbic acid as a precursor of serum complement. Nature 137-6 1 8 .

27. Morgan, E. S. 1936. Ascorbic acid as a precursor of serum complement. Nature 137*872.

2 8 . Chu, Fu-T1ang and B. F. Chow. 1938. Correlation between vitamin C content and complement titer of human blood plasma. Proc. Soc. Exp. Biol. Med. 38:679.

29. Ecker, E. E ., L . Pillemer, D . Wertheimer, and H. Gradis. 1938. Ascorbic acid and complement function. J . Immunol. 34:19*

30. Ecker, E. E. and L . Pillemer. 1939» Complement and ascorbic acid in human scurvy, an experimental study. J . Am. Med. Assn. 112:1449*

31. Ecker, E. E ., L . Pillemer, E. W. Martiensen, and D . Wertheimer. 1938* Complement activity as influ­ enced by certain chemical agents. J . Biol. Chem. 123:351*

32. Agnew, S., W. W. Spink, and 0 . Mickelsen. 1942. Ascorbic acid and immunity. II. The relation of ascorbic acid to guinea pig complement. J . Immunol. 44:297* 38

33- Spink 9 W o ¥. 9 S. Agnew, and 0. Mickelsen. 1942. Ascorbic acid and immunity. I. The relation of ascorbic acid to human complement. J . Immunol. 44:289. '

34. Crandon, J . H .^ C . C . Lund 9 and D . B . Dill. 1940. Experimental human scurvy. New Eng. J. M e d . 223 * 353.

35- Kodicek 9 E. and B . Traub. 1943• Complement activity and vitamin C . Biochem. J. 37 » 45 6 .

36. Rangam^ C. M., J . Chandra, and A. N. Ria Chowdhary. 1956o Complement values in experimentally induced deficiency states in guinea pigs. J . of the Indian Med. Assn. (Calcutta) 2 7 :20$.

37. Smith, G. H. and I. M. Wason. 1923. Serological factors of natural resistance in animals on a deficient diet. J . Immunol. 8:195*

38. Osborn, T. W. B. 1931. A study of the factors influencing the concentration of complement in the blood. Biochem. J . 25-2136.

39. Rose, S. B. and J . A. Kolmer. 1936. Complement studies on dogs during B-avitaminosis and anhydremia. J . Infect. Diseases 5 9 :1 2 6 .

40 e Wertman, K ., L . W . Smith, and W . M. O'Leary. 1953» The effects of vitamin deficiencies on some physiologic factors of importance in resistance to infection. I. Niacin-tryptophan deficiency. J. Immunol. 72:196. kl . Pruzansky, J . and A. E . Axelrod. 1955• Effect of B-complex deficiencies on rat serum complement. Proc. Soc. Exp. Biol. Med. 88:179.

. , . t 42. Kenney, M. A., L. Arnrich, E. Mar, and C. E. Roderick. 1965 - Influence of dietary protein on complement, properdin, and hemolysin in adult protein-depleted rats. J. Nutrition 85:213-

43. Weimer, H. E ., J. R. Godfrey, R. L . Meyers, and J. N. Miller. 1 9 6 3 . Effect of food restriction and realimentation on serum proteins: complement levels and electrophoretic pattern. J . Nutrition 8l:405. 39 44. Weimer 9 H. E . 9 J . N . Miller 9 R. L . Meyers 9 D . Baxter, D . M. Roberts, J . F. Godfrey, and C. M. Carpenter. 1964. The effect of tumor growth, nutritional stress, and inflammation on serum complement levels in the rat. Cancer Res. 24:847.

45 o Richar, W . and E. Breakell. 1959« Evaluation of an electronic particle counter for the counting of white blood cells. Am. J . Cl. Path. 3 1 :384.

46 o Crosby, W. H ., J . I. M u n n , and F. W. Furth. 1954. Standardizing a method for clinical hemoglobi- nometry. U . S. Armed Forces Med. J . 5:693 «

4? - Boutwell, J . H. 1 9 6 1 . Clinical Chemistry. Lea and Febiger, Philadelphia! 3^0 p.

48. Umbreit, W. ¥., R . H. Burris, and J. F . Stanffer. 1957° Manometric Techniques. Burgess Publishing Co., Minneapolis. 33# P •

49 - Gornall, A. G., C . J . Bardawill, and M. M. David. 1949• Determination of serum proteins by means of the biuret reaction. J . Biol. Chem. 177:751«

50 o Darter, J. A. 1953° Procedure for production of anti-sheep hemolysin. J . Lab. and Clin. Med. 4l: 653.

51. McKee, A. P. and W. S. Jeter» 1956. The demonstra­ tion of an antibody against complement. J . Immunol. 76:112.

52. Reid, M. E. and G. M. Briggs. 1953° Development of a semi-synthetic diet for young guinea pigs. J . Nutrition 51:3^1 .

53 • Reid, M. E. 1954. Nutritional studies with the guinea pig: B-vitamins other than pantothenic acid. Proc. Soc. Exp. Biol. Med. 8 5 :5^7«

54. Baker, H., 0. Frank, J . Fennelly, and C. Leevy. 1964. A method for assaying thiamine status in man and animals. A. J . Cl. Nutrition l4: 197°