AD-ARB 846 LETTERMAN ARMY INST OF RESEARCH SAN FRANCISCO CA F/B 6/8INVESTIOATION OF POSSIBLE ANTIVITAMIN B-6 PROPERTIES IN IRRAOIA--ETC(U)JUN 81 E L MCGOWN, C M LEWIS, A ROBLES

UNCLASSIFIED LAIR-87EEEEEEEl/hmEE

llEEllEEllEEEEEIEEIIIIIIEEEEIIIIIIIIIIIIIIlEEEEEEIIIIIEI*EEEEEEEEEEF,8

INSTITUTE REPORT NO. 87

'~INVESTIGATION OF POSSIBLE ANITIVITAMIN B-6

'~PROPERTIES IN IRRADIATION STERILIZED CHICKEN

EVELYN L. McGOWN, PhD0CAROLYN M. LEWIS, MSALADINO ROBLES, BSPAUL P. WARING, MSJAMES H. SKALA, PhDVIRGINIA L. GItDENGORIN, PhDHOWERDE E. SAUBERLICHf, PhD

DIVISION OF RESEARCH SUPPORT D% LET

JUNE 19818

Lum

LETTERMAN AWAY INSTITUTE OF RESEARCH PIRSIDIO Of SAN FRANCISCO CALIFORNIA 94129

Investigation of Possible Antivitamin B-6 Propertiesin Irradiation Sterilized Chicken--McGowen et al

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In conducting the research described in this report, the investigation adhered to the "Guide for the Care andUse of Laboratory Animals," as promulgated by the Committee on Revision of the Guide for LaboratoryAnimal Facilities and Care, Institute of Laborato', Animal Resources, National Research Council.

This material has been reviewed by Letterman Army Institute ofResearch and there is no objection to its presentation and/orpublication. The opinions or assertions contained herein are theprivate views of the author(s) and are not to be construed asofficial or as reflecting the views of rhe Department of the Armyor the Department of Defense. (AR 360-5)

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4. &*t-rW 90111oy5. T~h _j .. RO COVERED

/Investigation of Possible Antivitamin B-6 Prop- Final Kepwtk -erties in Irradiation Sterilized Chicken *Nov *79-Aug W80

6. PERFORMING ORG. REPOR ; M BE R

7. AUTHOR(a) 8. CONTRACT OR GRANT NUMBER(*)

E.L. McGown, PhD; C.M. Lewis, MS; A. Robles,BS; P.P. Waring, MS; J.H. Skala, PhD; V.L.Gildengorin, PhD; H.E. Sauberlich, PhD

9. PERFORMING ORGANIZATION NAME AND ADDRESS 1_0. -PROGRALEMEN P Z, TASKAREA a W "~S

Division or Research Support, Letterman Army Proj. No.(3MI611 2B02Institute of Research, PSF, CA 94129 Work Unit 177

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U.S. Army Medical Research and Development Co / Jun q 81mand, Fort Detrick, Frederick, MD 21701 NUMBER OF PAUS

M!L ONI * ODIun AG"M(Y MAME &-AnnaES( V 1. tatrolling Office) I5. SECURITY CLASS. (of this report)

Evelyn L./McGown Carolyn M./LewisAladino/Robles Paul P./Waring UNCLASSIFIEDIJames H./Skala .IS&. DECLASsIFIcATION/OWNGRADING

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IS. SUPPLEMENTARY NOTES

IS. KEY WORDS (Continue onr, evers sc ide If necesary and Identify by block number)

Irradiated chicken; anti-vitamin B-6; vitamin B-6; pyridoxine; pyridoxal phos-phate aspartate aminotransferase; alanine aminotransferase

20, ABSThAcr (Cnime an severes etDb N risrstar m~d idew*Uify by block numqber)

The purpose of the study was to determine whether irradiation (gamma or electron)or thermal processing of chicken produces factors which are antagonistic to vita-min B-6 in the diet of rats. (These methods of preservation all result in lowcevitamin B-6 contents relative to frozen chicken.) Male and female rats (156 eachwere made vitamin B-6 deficient by feeding a semi-purified diet devoid of vita-min B1-6. They were then repleted with various test diets co)ntailinjg chicken WIlL'had het'n preserved by one (if four methods: frozewn, iLh rrii;I I I V p to( (((- ( CuI rol

DD "0" 1473 EDITION OF IMO Nos IS OBSOLETE UNLSSFE

SECURITY CLASSIFICATION Of THIS PAGE (When rDate Entered)

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Itenm 20 (Cuiit)

or gamma irradiated. All repletion diets were supplemented to contain carefully

controlled (marginal or high) levels of vitamin B-6. Recovery rates were moni-

tored by growth (weight gain) and measurements of vitamin B-6-dependent bloodenzymes (plasma and red cell aspartate aminotransferase and alanine aminotrans-

ferase). No differences were observed in weight gain among the chicken-fedgroups. The enzyme responses of rats fed frozen, thermally processed or electrorirradiated chicken were similar. Responses of some of the enzymatic parameters

were slightly delayed in the groups fed gamma irradiated chicken at the marginal

vitamin level. No consistent differences were observed between any of the high

vitamin groups. If antivitamin B-6 factor is present in gamma irradiated

chicken, it is minimal, is detectable only under conditions of marginal vitamin

B-6 status, and is overcome by added dietary pyridoxine.

UNCLASSIFIED

SECURITY CLASSIFICATION OF THIS PAGE("en Data Pnr..Id)

ABSTRACT

The purpose of the study was to determine whether irradiation(gamma or electron) or thermal processing of chicken produces factorswhich are antagonistic to vitamin B-6 in the diet of rats. (Thesemethods of preservation all result in lowered vitamin B-6 contents rel-ative to frozen chicken.) Male and female rats (156 each) were madevitamin B-6 deficient by feeding a semi-purified diet devoid of vitaminB-6. They were then repleted with various test diets containing chickenwhich had been preserved by one of four methods: frozen, thermally pro-cessed, electron or gamma irradiated. All repletion diets were supple-mented to contain carefully controlled (marginal or high) levels ofvitamin B-6. Recovery rates were monitored by growth (weight gain) andmeasurements of vitamin B-6-dependent blood enzymes (plasma and redcell aspartate aminotransferase and alanine aminotransferase). No dif-ferences were observed in weight gain among the chicken-fed groups.The enzyme responses of rats fed frozen, thermally processed or elec-tron irradiated chicken were similar. Responses of some of the enzy-matic parameters were slightly delayed in the groups fed gammairradiated chicken at the marginal vitamin level. No consistent dif-ferences were observed between any of the high vitamin groups. If anantivitamin B-6 factor is present in gamma irradiated chicken, it isminimal, is detectable only under conditions of marginal vitamin B-6

status, and is overcome by added dietary pyridoxine.

ii

FOREWORD

Recent U.S. Army protocols for the wholesomeness testing of rad-appertized meats and poultry (1,2,) included antivitamin studies forthiamin and vitamin B-6. These studies were designed in response toquestions raised by the Food and Drug Administration (FDA) in 1968,after review of data in the readappertized ham petition. This fore-word provides a brief historical background that led to the require-ment for the antivitamin studies in the irradiated food protocols.

In 1953, the U.S. Army was assigned the task of determining thewholesomeness of foods preserved by radappertization (sterilizationby ionizing radiation) and of developing the process. Protocols weredesigned with the assistance of scientists from industry, universities,and FDA. These studies were essentially completed by about 1964. Ingeneral, the foods under study were canned and irradiated at ambienttemperature at two dose levels (2.79 and 5.58 mrad) with spent fuelrods. Radappertized foods were stored at room temperature and the con-trol canned foods were stored frozen.

Bacon was one of the most promising foods preserved by radapper-tization. In 1963, a petition for cobalt-60 radappertized bacon wasapproved by FDA (3). Subsequently, approval was obtained for irradi-ation with 5-10 MeV electrons, 5 MeV x-rays, and Cesium-137. With theapproval of the bacon petition, a petition for ham was submitted. Thispetition was based on bacon and pork data. Expert opinion was inagreement; since ham was intermediate in degree of processing to baconand pork, it did not need to be tested. After review of these data in1968, FDA concluded, in part, "Our evaluation of the raw data led usto believe that there were suggestions of adverse effects and that,therefore, the safety of these irradiated meats had not been established"(4p96).

Subsequently, the U.S. Army withdrew the ham petition and FDA re-scinded its previous approval of radappertized bacon (4pp123-124).Although some of the suggested adverse effects reported by FDA were amatter of opinion and interpretation, it is not the intent or purposeof this foreword to rebut them. However, it should be recognized thatthe pioneering U.S. Army irradiated food program was initiated beforethe Food Additives Amendment of 1958 and its later rigorous interpre-

tation and enforcement (4pp127-132).

Pertinent to the present issue, an adverse effect was observed

"apparent production of antinutrient factors" (4p104). This conclu-sion was based primarily on incomplete data in the progress reports of

Brins' group (5-7) in which blood transketolase and transaminase ac-

tivities were reduced in rats fed radappertized pork, unsupplementedwith thiamin or pyridoxine, respectively, and were not restored to nor-mal upon repletion with the respective vitamin (5,O). If the pork

iii

l-iECE1MQPAG iLI~-OT h)U

...I{ '

diets were supplemented initially with the vitamins, the enzyme activ-ities were not affected by the irradiated pork (5,7). Although thestudies might be questioned, this foreword will not discuss the meritsof the data or their interpretation by FDA; however, it would appearthat when the wholesomeness of radappertized foods is under considera-tion, the question, i.e., production of antinutrient factors, raised byFDA must be resolved to the satisfaction of all concerned.

Some persons question the value of the "old" wholesomeness studies.Irradiation technology has not only changed and improved, but also therequirements for additive testing have become more stringent. Sincethe present day radappertized products are superior to those first-generation products, it could be assumed that the earlier productspresented the worst possible case. Also, if any irradiation-inducedtoxicity had been present, it would have manifested itself moreprominently. In spite of this, when adverse effects were found, sub-sequent investigations disclosed that these were not caused by ir-radiation but resulted from other complicating factors. One suchproblem, which generally has been given inadequate consideration, wasthat radappertized foods (very highly processed and stored at ambienttemperature) have been (unfairly) compared to unprocessed frozen con-trols. Furthermore, the wholesomeness studies have attempted to provea negative -- virtually an impossible task.

Investigators (8,9) from Letterman Army Institute of Research havealready published two reports on thiamin and erythrocyte transketolaseactivity in which they used irradiated beef (8) and irradiated chicken(9). The following report describes the effects of dietary pyridoxinelevels and radappertized chicken on blood transaminase activities in

rats.

NICHOLAS RAICA, JR.ConsultantU.S. Army Medical Researchand Development Command

REFERENCFS

1. DEPARTMENT OF TI1F ARY. Animal Feeding Protocol for IrradiationSterilized Beef. Washington, DC: Research Directorate, U.S.Army Medical Research and Development Command, 1971

2. DTEPAPThIFN OF 71!r ARY. Animal Feeding Study Protocol for Trrsdi-ation Sterilized Test Foods. Washington, DC: Office for theWholesomeness of Irradiated Foods, 11.S. Arrv Medical Research andDevelopment Conmand, 1975

iv

3. FEDERAL REGISTER. (121.3001), p 1465, 1S February 1963

4. Status of the Food Irradiation Program. llearings before the Sub-committee of the Joint Committee on Atomic Fnergy, July 18 and30, 1968

S. OSTASIIEVER, A.S. H. BRIN, H. TAT, and H. KALINSKY. The effectof irradiated foods on specific enzyme levels in blood and intes-tinal tissue. Contract No. DA 49-007-M 862, 1AS7-1QS9

6. BRIN, H., A.S. OSTAS1EVER, H. TAT, and HI. KALINSVY. Effects ocfeeding x-irradiated pork to rats on their thianin nutrition asreflected in the activity of erythrocyte transketolase. T Ntutr75:29-34 1961

7. BRIN, H., A.S. OSTASHFEVERP, M. TAT, and 11. KATNSKY. The effectsof feeding irradiated pork, bread, green beans and shrimp tn rntson growth and five enzymes in blood. Toxicol Appl PhMrracol 3:606-617 1961

8. MCGOWN, I.I,., C.11. LEWIS, and P.P. WARINGC. Investigation of os-sible Antithiamin Properties in Irradiation Sterilized Peef.Report No. 71. San Francisco, California: Letterman Arv Insti-tute of Research, August 1979

9. MCGOWN, E.L., C.M. LEWIS, and P.P. 1VART'1G. Investigation o Pos-sible Antithiamin Properties in Irradiation Sterilized Chicken.Report No. 72. San Francisco, California: Letternan Army Insti-tute of Research, August 1979

v

PREFACE

The experimental portions of the study covered in this report wereconducted during the period 1 November 1979-1 June 1980. All raw dataare being stored at Letterman Army Institute of Research. Anyone wish-ing to examine the raw data or to obtain copies of tables containingindividual values may do so by contacting:

Commander

Letterman Army Institute of ResearchATTN: SGRD-ULZPresidio of San Francisco, California 94129

In addition to personnel listed on pagexithe authors gratefully

acknowledge the assistance of COL Ronald Johnson (MOBDES) who helped in-

terpret the data and prepare the report, and Ann Wilkinson and CarolAllen, typists. We particularly thank Lottie Applewhite, LAIR Technical

Editor, for her editorial assistance and for detecting several poten-

tially serious omissions.

vi

vii

-di- -

TABLE OF CONTENTS

Page

Abstract ............ ............................ i i

Foreword ............ ............................ .

Preface ............ ............................. .vii

Table of Contents .......... ........................ ... ix

Report of Quality Assurance Officer ..... ............... ... xi

List of Personnel .......... ........................ .. xii

Signatures of Principal Scientists ...... ............... .xiii

BODY OF REPORT

INTRODUCTION ............ ......................... 1

Background and Experimental Design ...... ............. 1

Pilot Study ............ ......................... 3

METHODS ............. ............................ 3

Chicken Test Meats .......... ..................... 3

Animal Care ............ ......................... 4

Diet Preparations .......... ...................... 4

Blood Sampling and Analyses ........ ................. 4

Data Acquisition and Handling ....... ................ 5

Statistical Analysis and Enzymatic Data ..... ........... 6

RESULTS ............. ............................ 7

Vitamin B-6 Analyses .......... .................... 7

Animal Body Weight Responses ........ ................ 7

Erythrocyte Aspartate Aminotransferase ..... ........... 8

Plasma Aspartate Aminotransferase. . ............. 9

ix

k-,MZMW pAGS UMm-OT I IJU6,t

Page

Plasma Alanine Aminotransferase .............. 10

Erythrocyte Alanine Aminotransferase ... ............ ... 11

DISCUSSION ........ ...................... ... 12

Comments Concerning Sensitivity of Experimental Parameters. 12

Aminotransferases as Indicators of Vitamin B-6 Status. . .. 12

Vitamin B-6 Assay ......... ...................... .14

Effects of Diet on Enzymatic Parameters ... ........... ... 15

CONCLUSIONS .......... ........................... 16

RECOMMENDATIONS ........ ............................ 17

REFERENCES ........... .......................... 18

APPENDICES

Appendix A (Figures I through 22) ....... .............. 21

Appendix B (Tables I through 17) .... ................... 45

OFFTCTAL DISTRIBUTION LIST ...... .................. 64

OFFICIAL COOPERATING AGENCIES ........ ................ 65

x

DEPARTMENT OF THE ARMYLETTERMAN ARMY INSTITUTE OF RESEARCH

To PRESIOIO OF SAN FRANCISCO, CALIFORNIA 94129

ATEN TION OF: >uor

MEMORANDUM FOR RECORD

SUBJECT: Report of GLP Compliance

I hereby certify that in relation to LAIR GLP study -'qO02 the folliewinr-inspec'tions were made:

20 December 1q'992 January 1980

13 January 1980I293 February 198029 Mear 198012 March 1980

Findings were reported to the Study Director and laboratory nanagemen',on It February 1980. Routine inspections with no adverse finlings nr-reported quarterly, thus these inspections are also included 4 n the 'Iy1980 report to management and the Study Director.

O08N L. SZUREKAJ, MS

Quality Assurance Officer

xi

LIST OF PERSONNEL

Study DirectorI.TC John T. Fruin

AdvisorDr. lHowerde E. Sauberlich

Principal investigators and Supervisory PersonnelDr. Evelyn McGown (Toxicology Group)Ms. Carolyn M. Lewis (Toxicology Group)SFC Aladino Robles (Animal Resources Group)Dr. James H. Skala (Analytical Chemistry Group)Mr. Paul taring (Analytical Chemistry Group)

Animal Care and Feed PreparationSP5 Ronald ThompsonSP4 Charlotte SpcckmanMr. Wanless HatcherPFC Robert J. Hughes

Microbiological AssaysMr. Peter Taylor

Laboratory SupportSP5 Michael C. RusnakMs. Mary F. LyonMr. John J. KnudsenSP5 Joseph Alletto

Computer ProgrammingMr. William 11. Langley, Jr.

Computer GraphicsMr. John J. Knudsen

StatisticianDr. Virginia L. Gildengorin

Civilian Consultant

Dr. Nicholas Raica, Jr.Denver, CO

U.S. Army Medical Research and Development CommandLiaison Advisory Officer

LTC Duane E. Hilmas/MAJ Frank ChappleChief, Office for the Wholesomeness of Irradiated Foods

Fort Detrick, MD

xii

Signatures of Principal ScientistsInvolved in the Study

We, the undersigned, believe the study described in this reportto be scientifically sound and the results and interpretations to bevalid. The study was conducted to comply to the best of our abilitywith the Good Laboratory Practice Regulations for Nonclinical Labora-tory Studies outlined by the Food and Drug Administration.

EVELYN L. $GOWN, PhD PAUL P. WARING, BS

Research Chemist Research Chemist

CAROLYN. LEWIS, MS ALADINO ROBLES, BS, SFCNutritional Scientist NCOIC, Animal Resources Group

^S H. SKALA, PhD VIRGINAi.L. GILDENGORIN, Phil)(Research Chemist Mathematical Statistician

HOWERDE E. SAUBERLICH, PhD JO T. FRUIN, DVM, PhD, LTC, VCChief, Division of Nutrition Study DirectorTechnology

xiii

kp

INTRODUCTION

The testing of control and irradiated meats for antimetaholiteactivity against vitamins B-I and B-6 is a requirement of the protocolentitled "Animal Feeding Protocol for Irradiation Sterilized TestFoods" originated by the Office for Wholesomeness of Irradiated Foods,U.S. Army Medical Research and Development Command (USAMRDC), dated21 October 1975.

The purpose of the study reported here was to determine whetherirradiation (gamma or electron) or thermal processing of chicken pro-duces factors which are antagonistic to vitamin B-6 in the diet ofrats.

Background and Experimental Design

The present study is similar to another antivitamin study whichwas conducted at this institute (1). The same lot of chicken was usedand many of the standard operating procedures were identical. Someof the experimental details common to both studies have already beendescribed (1) and will be cited rather than repeated in this report.

The protocol for the antivitamin B-6 study specified that ratswere to be made deficient in vitamin B-6 (according to a pre-set weightgain criterion). They were then to be repleted with various chicken-containing or semi-purified diets which had identical (high or mar-ginal) vitamin B-6 contents and the recovery rates were monitored. Adecreased recovery rate in animals fed irradiated meat relative tothose fed control meat could indicate the presence of an antivitaminB-6 substance. Such a substance could arise (but not necessarily)from radiolysis of endogenous vitamin B-6.

Resumption of weight gain was the obvious indicator of recovery.The other parameters specified by the protocol were blood transami-nases which require pyridoxal phosphate for activity: serum aspartateaminotransferase (AST), serum alanine aminotransferase (ALT) anderythrocyte AST. These enzymes are decreased in vitamin B-6-deficientrats and humans (2,3). Transaminase activities are much lower in

serum than in erythrocytes and the serum enzylilec; are not us i . r, drelitable indicators of vitamin B-6 status, at leaist in tilt, hu:.iii ,4)in fact, serum AST measurements are commonly usedI to detoLvCt u,1 path.J-logical conditions as myocardial infarction rather thun to asstssnutritional status. Lrythrocyte AST measurements a,,d, in partlctlzir,in vitro stimulation by pyridoxal phosphate are cet.idcrd t, be thcmost useful methods for assessing vitamin B-6 S. tatus ki.34). (.rvtII-rocyte ALT has not been a common measurement beci.use f 1,11 j jdifficul ty of assaying the low activity in the presence -I 1m. ..bin in hemolvsaites.) The "activi.ty coefficc ct" ratio ,f -t i i tedto unstimulated activity is presumably indicativc of the proportion j!enzyme not s_,aturated with cofactor.

The protocol specified that the neat diets contain 35Z:J test iat(on a dry weight basis). Furthermore, it specified that each nmea.t betested at two levels of vitamin intake, a marginal level and a highlevel. The high vitamin diets were included to determine whether ornot any antivitamin B-6 substance (if detected) could be overcome byadditional vitamin.

All test meats originated from one lot of chicken which had beenheated to inactivate enzymes. This lot was divided into four partsand each was further prepared for storage by one of the follo,'ingtreatments: frozen (control); canned (thermally processed); F,,iiini(cobalt) irradiated; electron irradiated. The last three treatmentsproduce shelf-stable products and are known to cause decreased vitamincontent. Finally, the protocol specified the inclusion of groups feddry semipurified diets without chicken.

To conduct a successful and scientifically valid antivitamin B-6study according to the guidelines of the protocol, we found it neces-sary to solve several problems before undertaking the study. Thevitamin B-6 microbiological assay posed particularly challengin., prob-lems which will be described later in the Discussion. Another problemwas the lack of a reliable standardized erythrocyte transaminase as-say which would be suitable for large numbers of samples. Al thoughthe serum assays had beer well established (in fact, commercial kitsare available), erythrocyte transaminase assays left much to he de-sired (3). To remedy this situation, an autoanalyzer procedure wasdeveloped to assay erythrocyte AST in washed red cells and a Gemsaecfast analyzer method for whole blood AST was developed. Since w,,could not know in advance which method would prove more valuable,both assays were done on all samples. Activity coefficients on1 theplasmax enzymes were also determined, although this was not specifiedby the protocol.

A third problem was the fact that, the protocol specified mar-ginal repLetion levels to be 8.0 mg pyridoxine/kg diet, but wesuspected that this level might not be low enough to provide a sensi-Live Lest system. Some reports have suggested that ti B-0 requirement

2jLk5

%t

of the rat is lower than 8 mg/kg (5,6). Lastly, we felt it essentialto establish that repeated bleeding of rats would not cause elevatedserum transaminases due to muscle damage during cardiac puncture. Toanswer the two latter questions and to establish the validity of theerythrocyte AST assays, we conducted a pilot study.

Pilot Study

Twenty-four female weanling rats were made vitamin B-6 deficientas indicated in the Methods. A blood sample was taken from each fortransaminase analyses and the animals were divided into 3 repletiongroups of 8 each. The casein-based repletion diets contained 2.0,

4.0, and 12.0 mg pyridoxine'HCl/kg diet. A group of 6 non-deficientcontrol rats was maintained on the 12.0 mg/kg diet throughout thepilot study. Figure 1 shows the growth curves of the four groups dur-ing the repletion phase. All three repletion groups immediately ex-hibited striking weight gains. After 4 weeks, there was a small butsignificant difference in mean weights between the lowest and highestvitamin groups. There was little difference between the 4.0 and12.0 mg/kg groups.

The erythrocyte aspartate aminotransferase data yielded similarconclusions. Within 7 days, both enzymatic activity and activity co-efficients had returned to their normal ranges except in the lowestvitamin group (Figure 2). Therefore, it appeared that in order topromote marginal recovery rates, the diets had to contain less than4.0 mg pyridoxine/kg diet and 2.0 mg/kg appeared to be suitable. How-ever, endogenous vitamin B-6 in the frozen control chicken (at 35%dry weight) contributed more than 2 mg/kg to the diet (Table 1). We,therefore, chose 2.5 mg pyridoxine'HCl/kg diet to be the marginal re-pletion level.

The final question addressed in the pilot study was whether re-peated blood samples could safely be taken by cardiac puncture. Wefound no evidence that cardiac puncture caused elevated serum trans-aminases within the time frame which would interfere with our study(data not shown). Although an occasional sample did have elevatedvalues, this occurred so seldom that we did not feel it posed a threatto the study.

METHODS

Chicken Test Meats

All test meats were supplied by the U.S. Army Natick Research andDevelopment Command, Natick, MA. They were from a lot labelled "Lot3" and "Lot 3A" and had been processed according to the procedure out-lined in Appendix A of the protocol described in paragraph 1 of pageI. The enzyme inactivation, gamma irradiation and electron irradia-tion have been summarized in our previous report (1).

3

-. P

Animal Care

Male and female weanling rats (156 each) were purchased fromCharles River Breeding Laboratories, Wilmington, MA. Each anima'l wasidentified by ear tag and individually caged in a room with a 12-hrlight/dark cycle. All were given ad libitum water and a semi-purifieddiet containing 12.0 mg pyridoxine'HCl/kg diet. The casein-based dietwas the same as described previously (1) except a commercial salt mix-ture was used (Rogers & Harper salt mix (7), purchased from ICN Nutri-tional Biochemicals, Cleveland, OH, and added as 4% of the diet).

The schedule and diet codes for the two studies are outlined inTable 2. After one week of quarantine and adaptation (Phase 1), 24 ratswere selected at random to remain on diet A. All other animals wereplaced on diet B, which was identical to diet A, except that pyridox-ine had been omitted (Phase 2). Growth was monitored throughout thestudy by thrice weekly weighings. Animals on diet B were consideredto be deficient when the average daily weight gain was less the 1.0 g.The deficient animals were then randomly divided into 11 groups of12 animals each. The diet A animals were also divided into 2 1groupsof 12 each. One group of 12 diet A rats and one group of 12 diet Brats were bled by cardiac puncture and removed from the study. Theremaining 10 groups of deficient animals were placed on 10 differentrepletion diets (C through L) and the remaining A group was continuedon diet A. The repletion period (Phase 3) lasted 4 weeks.

Lie t_l'ipjalrat i ons

Values tor proximate analyses, calcium, and phosphorus were re-ported previously (1). Vitamin B-6 assays were done by a microbiolog-ical method which utilizes Saccharomyces uvarum (formerly S. carisber-&etis) a, the test organism (8).

As specified by the protocol, the meat diets were formulated tocontain 35% (dry weight) chicken. The fat and protein levels of thesemi-purified diets were adjusted to be similar to the meat diet.,based on calculations from proximate analysis data (1). For each ofthe meat treatment groups (E through L), a dry premix with fat and pro-tein omitted and containing the proper amount of pyridoxine-llC1 wasprepared in advance. When mixed with the corresponding meat (35% dryweight basis), the complete diets contained the specified levels ofvitamin B-6 and were similar to the semi-purified diets, except thatground chicken replaced the casein, lard, and corn oil. Furthir de-tails of the mixing and feeding procedures were included in the pre-vious report (1).

Blood SampjtnG and Analyses

Blood samples were obtained by cardiac puncture from all rats ondays 7, 14, and 28. The animals were anesthetized with penthlrane gas

4

and samples (2.5 ml each) were collected into EDTA-containing syringes.Hematocrits were determined in duplicate on each sample by the micro-capillary centrifugation method. Aliquots of each were centrifugedand the plasmas were removed (in subdued light) for AST and ALT assays(done on the same day as the blood drawing). The red cells were washedtwice with saline, hemolyzed with distilled water, and stored frozenuntil erythrocyte AST assays were done.

AST activity in sera and whole blood hemolysates (18 X dilutionof previously frozen whole blood with 0.9% saline containing 0.1%Triton X-100) was determined with a Gemsaec Clinical Analyser. Thechemistry employed was a slight modification of the method recommendedprovisionally by the International Federation of Clinical Chemists(IFCC) (9); the principal modifications consisted of lower concentra-tions of substrates and lower coupled enzyme activities, lower sampleto total reactiom volume ratio, and initiation of the reaction withsample rather than reagent. Serum ALT activity was determined usingsimilar modifications of the chemistry recommended by the ScandinavianSociety for Clinical Chemistry and Clinical Pathology (SSCP) (10).The details of these methods have been published in a handbook of vita-min B-6 methodology (11). Activities were determined with and withoutexogenous pyridoxal-5'-phosphate.

Erythrocyte AST (EC 2.6.1.1.) was measured by an adaptation of themethod described in Technicon Method SE4-0010 FH4 (12) for serum analy-ses, based on the work of Kessler et al (13). The reagent concentra-tions were adjusted to permit optimization of the enzyme reactions asrecommended by the IFCC (9,14). The apparatus was modified to allowthe semi-automated measurement of pyridoxal phosphate-stimulated ASTactivity under system-controlled reaction conditions. These modifica-tions are described elsewhere (11).

Although erythrocyte ALT measurements had not been specified inthe protocol, inspection of erythrocyte AST and plasma ALT data sug-gested that erythrocyte ALT data might be useful. Therefore, an auto-analyzer assay was developed and the erythrocyte samples were reassayedfor ALT (two months after the original AST analyses). The procedurefor the measurement of erythrocyte ALT (EC 2.6.1.2.) was based on themethod recommended by the SSCP (14) as described in Sigma Tech. Bul-letin No. 57-UV (7-79) (15) for the analysis of serum. Reagent rela-tionships were modified as required to adapt the procedure for use withthe continuous flow analytical technique for the Technicon AutoAnalyzerII. Duplicate analyses for erythrocyte ALT activity were performed inthe presence and absence of pyridoxal phosphate to derive activity co-efficients.

Data Acquisition and Handling

Animals were weighed using an electronic balance interfaced witha programmable calculator. The weight data were permanently printed on

paper tape and recorded on a miniature tape cartridge. The informa-tion on the magnetic tape was then transferred to a minicomputer(Data General Eclipse C330) to be processed and released in reportformat. Programs for transferring and processing the data were de-veloped locally in the Information Sciences Group.

Plasma aminotransferase data were expressed as IU/liter on theprint-out associated with the Gemsaec Fast Analyzer. These data weremanually transferred to code sheets and card punched. Erythrocyteaminotransferase data were recorded in digital printout form as "chartunits" on paper tape from the autoanalyzer. These data, appropriateconversion factors, and hematocrit values were also manually enteredonto keypunch coding sheets. Calculations of enzymatic activity interms of [U/ml red cells were done by the computer (programming bythe statistician) in conjunction with the statistical analyses.

Quality control data for both plasma and erythrocyte aminotrans-ferase activities are summarized in Table 3.

Statistical Analysis of Aminotransferase Data

Past experience has demonstrated the effects of diet and level ofvitamin B-6 to be different for the different sex groups. Hencc, themeasurements taken on the three sample days (7, 14, and 28) were ana-lyzed separately for each sex to determine the main effects of thedesign variable (i.e., diet and level of vitamin B-6) along with theirinteractions on each aminotransferase and its activity coefficient.

Based on the assumptions of normality, statistical independenceand the equality of subgroup variances, two-way analyses of varianceswere performed with a packaged computer program, BMP Biomedical Com-puter Program P2V (16).

The following model was used in the two way analyses of variances:

y = m + a. + b. + ab.o + e1 j IJ

where y = observed enzymatic activity

m = overall mean effect

a. = effect due to diet group

h. = effect due to vitamin levelJ

ab.. = effect due to diet by vitamin level interaction1J

e = error term

6

When the results of the analysis of variance gave a significantF value for diet effects, a posteriori multiple comparisons were usedto test for differences among means by the Newman-Keuls procedure (17).In some of the multiple comparisons, the Newman-Keuls test was unableto find significant group differences and therefore, 95% confidenceintervals were also examined. Three cases were found in which analy-sis of variance (ANOVA) suggested diet effects, but neither Newman-Keuls nor the confidence intervals revealed any diet differences withinthe vitamin levels. The variances in these cases were significantlydifferent by Bartlett's test procedure (17). Therefore, the Kruskal-Wallis one-way analysis of variance procedure (18) was used separatelyon each vitamin group in these three cases to test group differences.Differences were found in two of them and these have been noted inthe ANOVA summaries.

RESULTS

Vitamin B-6 Analyses

Results of vitamin B-6 analyses in the chicken meats are presentedin Table 1. Frozen control chicken had the highest vitamin B-6 con-tent. Thermally processed, electron irradiated, and gamma irradiatedhad 83%, 63%, and 50%, respectively, of the frozen level. Table 1also includes the calculated amounts of vitamin B-6 contributed bythe chicken in the diets as well as the amount of crystalline pyridox-ine added to each to obtain the desired total (2.5 or 12.0 mg/kg). Itis obvious that most of the vitamin B-6 in the marginal frozen andthermal chicken diets had originated in the meats. In contrast, approx-imately half of the vitamin in marginal gamma and electron diets wasexogenous pyridoxine.

The vitamin B-6 contents of the test diets as determined bymicrobiological assays are also summarized in Table 1. Among themarginal diets, the frozen control consistently assayed slightlyhigher than the other groups, despite the fact that the diets had beencarefully formulated to contain identical amounts. The reasons forthe apparent discrepancy will be discussed later.

Animal Body Weight Responses

Growth (weight) curves for both males and females during thequarantine and depletion phases are shown in Figure 3. Non-deficientand deficient groups had similar weight changes for 10 days, afterwhich growth slowed considerably for both males and females on thedeficient diets. The criterion for deficiency of weight gains lessthan 1.0 gram/day was met at 33 and 42 days for females and males, re-

spectively. Because males required longer to reach this state of de-ficiency, their body weights were much greater than the females at

the beginning of the repletion phase.

7

Growth curves for the rats fed the repletion diets are pre;eu ciin Figures 4-8. In each case, the growth curve for the non-deficientcontrol group is shown to simplify the comparisons. Apparent jogs inthe curves occHIrred :ifter 7 and 14-day bleedings, presumably becaoseof physiological stress on the rats. Occasional animals died follow-ing bleeding days, but we included their body weights with the datarecorded prior to death.

ihen all groups are compared, it is obvious that rats consumingmeat diets regained their weight faster than those fed the semi-puri-fied repletion diets. This could be attributed to the fact that therats fed meat diets ate more feed (on a dry-weight basis) than thosefed the semi-purified diets (data not presented), presumably becausethe meat diets were more palatable.

When fed meat diets, females regained their weight and caught upwith the non-deficiunt controls by or before the end of the repletionphase. In contrast, although all male groups exhibited marked recoveryof weight, none of them reached the weight of the non-deficient con-trol group by day 28 of repletion. (This was probably due to thelarger difference between controls and deficients after the longerdepletion phase.) Neither males nor females recovered completely whenfed the semi-purified repletion diets.

Means for initial and final weights for the repletion phase andaverage daily gains calculated by week and for the total 28 days areshown in Tables 4 and 5 for males and females, respectively. Inspec-tion of these data, as well as Figures 4-8, reveal no differences be-tween the marginal and high vitamin groups. Thus the level of2.5 mg pyridoxine per kg diet apparently satisfied the repletion re-quirement for vitamin B-6 as far as growth criteria are concerned.Furthermore, no differences appeared between any of the meat groups,

although all of them supported better gains than the semi-purifiedreplet iou diets.

Frythmtl royt Aspartrte Aminiotrans ferasc (AST)

Analysis of variance significance levels for erythrocyte \SI arepresented in Table o. Both enzymatic activity (unstimulated) and ac-tivity coefficients were sensitive to vitamin B-(, intake, as revealedby the highly significant and consistent P values in the vitamin ef-fect column. Significant diet effects (P < .05) were detected forenzymatic activity in 4 out of 6 sampling clays and on all days foractivity coefficients. The sources of these treatment effects ,ill 1Cexamined later.

[rythrocvte AST group means are graphically summarized in Figures9 (male.) and 1o (females). Before repletion, erVthrocy te AS' in tihcdeficient males and females was 25% and 40'0, respectively, of thelevels in the corresponding non-deficient controls. Within 7 days,

. . ... . . . , 2

all repletion groups were well above deficient levels and by day 14,

they were at or above the levels of the non-deficient animals. For

all practical purposes, one might consider all groups to have re-

covered within 2 weeks when compared to animals which had never been

deficient. However, the high and marginal vitamin repletion groupsremain clearly distinguishable through 28 days.

Activity coefficient results are summarized in Figures 11 (males)and 12 (females). At day 0, the deficient groups had mean activitycoefficients of 1.94 (males) and 1.68 (females) compared to 1.06 for

non-deficient animals (both sexes). By day 7, all groups were wellbelow the deficient values, and by day 14, were at or near the non-

deficient levels. In agreement with the enzymatic activity resultsdescribed above, the effect of vitamin intake was obvious through 28

days: all high and low vitamin lines in Figures 11 and 12 are clearly

separated. The only obvious effect of diet was the tendency for the

semi-purified (marginal) group to recover more slowly.

Tables 7 and 8 give group mean values and the results of the

Newman-Keuls multiple comparison tests. Some significant group dif-

ferences were detected, but none was consistently observed throughout

all sampling days. The semi-purified group was slowest to recover

(lowest activity and highest coefficients during the first 2 weeks).

The gamma irradiated groups tended to recover slower than the frozen

controls, but this was statistically significant for enzymatic activ-

ity only on day 7 (males), and for activity coefficient on days 7 and

28 (both sexes). The groups fed electron irradiated chicken fared as

well or better than the frozen control group.

Plasma Aspartate Aminotransferase

Analyses of variance significance levels for plasma AST are sum-

marized in Table 9. Plasma AST was less sensitive to vitamin intake

than the erythrocyte enzyme and P values were significant only on day

7. However, the activity coefficient was significantly (P < .05) af-fected by vitamin level in all cases except day 28 for males.

Graphic comparisons of plasma AST group means are shown in Fig-

ures 13 (males) and 14 (females). At day 0, the deficient animals

had activities of 38% (males) and 35% (females) of their respective

non-deficient controls. By day 7 of repletion, all groups were nearly

back to non-deficient levels. The effect of vitamin intake (high vs.

marginal) was obvious for all treatments on day 7 (males), for irradi-

ated treatment on day 7 (females), and for frozen, thermal, and gamma

treatments on day 28 (males). On other days, there was no clear-cut

separation between high and low vitamin groups.

Activity coefficient group means are graphed in Figures 15 (male)

and 16 (females). At day 0, the deficient animals had activity co-efficients of 2.5 (males) and 2.7 (females) compared to 1.2 and 1.3

9

for respect ive non-del ic ent groups. By day 7 , itIgop a re,-turned to, non-deficient levels or nearly SO . .\ tho0ugh tilie ma~rgiralvitamin groups tended to have hithIer activation coefficiori t:; thanl th'ehigh v it ai il gro upq, this was not consistent except (lil days 7 -ind 1 *4

in the males.

Plasma AST group meanls anld roLSo Its ,tNI.'iaiie~anttpccm

pari-son tests are summairi.; ed inl Tab Les 10 (AST activitiv) and 11 (,,ic-tiVi ty coot iCitIS ) . meeorit~ ians wor i ire eI.dittel-Lcro(P < .05) but no cons istent trend was obs - rved . T[he g-roups tuod g iai

irrad iated chicken tended to rocover slower than those reec Lvintg I. ho-zen chicken. Trhis trend was significant only onl day 7 for males ;111on davs 14 aind 218 for foia les (act:Lvity coefific LentL only)'I. III a <,reu-ment with erythirocyte AST parameters, thle elkoetron-irradijited g-roupswere not dif ferent froom the frozen cont. t i gr)ups

Plasma Alanine Amiinot rans lerase

Analysis of variance significance levelS for plasrma Al.; and a:,-tivity coefficient are presented in 'Table 12. Both paramevons werehighly sensitive to vitamin intake as indicaIted b%, thle low P vailuesin the vitamin effects column. Somne effect-; Of diet WoeL 'Otcetedand these will be examined later.

Grapice summa ries of p lasmia ALP glroup means a re shown in l'ist.'tlr17 (males) andL 18 (teniales) . At day 0, titl' act li ties ill p ISlatiotdeI icIi en t ata Il s .;We I-r'o 8,, (ma les ) 'ind I -)k, ( tonal e-; )of thei( rec-1 ,t Le

cont rolI vailuc s. PyIV 7 tot r'p)itlioo, the Inargi ott I groups h-ie Li-creased to nea rly thle !LeVels Of non-dief ietL Z~tr l ad tiIL le higvitamin groups, hald atllvsurpas sed them. [he- ili gh vi1 alm ilo gratlpsremai ned elevated thlrouuhOut the 4-week observe t Lou period. Alolngwith this dratmatic and persistent OVershoot ofI :alismia ALl , tia iihand marginal Vitamin rep letion groups wore, 01 ar iv dit f ercnia ~ted at.a1 I I t I 11105. [ he Oafn ttd 0 he Sejiaral t i On asgreateCr ti1tan had ),1eIo)Ln2ohservcd Wi th the pli] sma or erChlrot-vte AS I . Ater L te 7- ,. ti we , ,li ttle further imp toYvhtLl t. ot the mri aIvi tam111 n I rati s in cithcirsex withI respect to t he no n-defI c ie n t c on t ro I s o, I t he Il I I\ it at mi in

groups . The only apparent effec t of diet was the tendec for tilefrozen (marginal ) group to have hi gheLr enzyma tic lctiv it v t han theother warginal groups.

P1 tisma ALT act ivit ' v coot f i .it cots areo summatitzed graphilcallyv inlFigures 19 (males) and 20 (females) . lie 0Ot i talest ha,12S1,d 'I"t I v i ty

coefficients of 2.0) and females 2. i comparotid to 1 . 10r lion-dc fi ci ut.animals (hoth1 sixe's) onl d ay 0. By day 7, a 1I I ij vi 1 11ii lO vplet iongroups had dr-opped to tlon-do ticijent levels Or1 bL'1OW. ;\I I II0aiI a IImarginal replttion groups had strikingly improl~ved I) dilv 7, thekir

aLiVIV ckcd tienLus remained elevated and Ii st inLe Itail Lilt, highvi tamin rep let ion groups throughout the 4-week oh so yi'i jil per iod.There was lit0 +viou e~Sf feet of diet except, tehpilhe faIster

Ito

recovery of the males fed frozen chicken (marginal vitamin). Althoughdeficient females had higher activity coefficients, they recoveredfaster and more consistently than did the males.

Plasma ALT group means and results of Newman-Kuels comparisonsare summarized in Tables 13 (enzymatic activity) and 14 (activity co-efficients). Few effects of diet were detected, and whenever differ-ences were found, the frozen group always had highest enzymatic activ-ity and lowest coefficients. The gamma-fed males did differ signifi-cantly from the frozen group on days 7 and 14 (both parameters).However, this was not observed in the females except on day 7, andthen only in enzymatic activity (Table 13).

Erythrocyte Alanine Aminotransferase

Because of the high sensitivity and reliability of plasma ALT asan indicator of vitamin B-6 intake, we felt it would be worthwhile toexamine tile erythrocyte enzyme. To do so required development of ananalytical method. We adapted the autoanalyzer method for erythrocyteAST and improved the sensitivity to accommodate the lower ALT activityin the erythrocyte (approximately 20% of AST levels). The assays weredone on the samples which had been assayed originally for erythrocyteAST. By the time the ALT assay could be done, the samples had beenstored frozen (-700C) for approximately 3 months. We have since foundALT to be stable for at least 3 months when hemolysates were storedunder these conditions (data not shown).

Analysis of variance significance levels for erythrocyte ALT' andactivity coefficients are given in Table 15. Both parameters werehighly sensitive to vitamin intake except the activity :oefficient onday 28 in the females. Many significant effects of diet and diet-vitamin interactions were detected which Till be examined below.

Graphic summaries of erythrocyte ALT group means are shown inFigures 21 (males) and 22 (females). At day 0, the deficient groupshad 12% (males) and 20% (females) of the activity of the non-deficientcontrols. During the four weeks of repletion, progressive increaseswere observed in all groups. The high vitamin groups increased faster,but had not quite returned to non-deficient levels by 4 weeks of re-pletion. The only consistent effect of diet was the fact that at alltime points, the semi-purified marginal group had the lowest groupmean.

Erythrocyte ALT group means and results of Newman-Keuls compari-sons are summarized in Tables 16 (enzymatic activity) and 17 (activ-ity coefficients). Among the marginal groups, the semi-purified con-sistently had the lowest activity and the frozen group the highest(except for the unusually high electron mean on day 7 in the males).The marginal gamma group means were lower than the marginal frozen,but were significantly so only on days 14 and 28 (both sexes). The

"" It . ... ...... ... Mlllll .... .. "' ' '"-' .... ''r-'' ' ''' "

... ; ........ ..... "- ..... ......... ,..... -

Ii I g;mml Ir,,I;) W,i ls :Is igi icaintl\' lower u1! dai, 7 ( ,E i t,:) 2 :day 28 (bat I -xcs). L ,'Lron rradiated was s i ,.:i FlcantL 1 % -( , r t! ll:1the frozen control in 4 Oat o! 0 compe Isons of th, ma r;, i a 'I L :iw

level and slightly, but significantly lower in 3 of the r .umparisonsat the high vitamin I eve]

Activity coefficients of erytLhroc-,te ALI ; ,reasud -ir dr: ivitamin B-6 deficieni-y than any other activity coei-fi-i-ttts. A, !0, t lie de fic ient groups had activity coef Icients of 1 .27 (mals -) and1. 16 (females) compared to 1.06 and 1.05 for the rospetive nor-dt i-cient groups (f,,otnote. Table 17) . Despite these sMa]] aI bs lutedifferences, ANOVA h;id revealed this parameter to bc sensitivc 'o ViLO-min intake on all sample days except day 28 in the females (Table 15).Significant differt.nces were detected between diet groups Py .iewinan-Keuls only on days 14 and 28 in the males (marginal vitamin Level);the frozen group had tile lowest, and the semi-pur ified, the highestactivity coefficients. However, the importaace of these differencesis doubtful because all means were at or below the level -f tle non-deficient controls.

DISCUSSION

Comments Concerning Sensitivity of Experimental Parameters

The sensitivity of our test system was limited by the stipulationin the protocol that the test meats be fed at 357 of dry weight of Liicdiet. At this level, the meats contributed to the diets 1.2 to 2..; nz

vitamin B-6 per kg diet. Therefore, the marginal repletion l,, - inthe study could not be less than 2.4 mg/kg of diet. in the pilot stud\we lad found 2.0 but not 4.0 mg/kg to be suboptimal relat!',,e Co 12.0 ra/kg. The low vitamin level selected in the major study (2. mg/ikg) Sup-ported growth responses which were no different than repcooi.i- to12.0 mg/kg of diet. However, the low vitamin level was truly marginalas revealed by tbe distinctly slower recovery rates of all enziyme pa-rameters.

Vitamin B-6-dependent processes involved in growth (weizht gain)took priority over the blood aminotransferases during repletion,. Thelow vitamin diets contained enough pyridoxine to support maxim-.a] orowthresponse, but recovery rates of the aminotransferases were clearlyslower when compared to the high vitamin groups. Thus the blood enzymeswere more sensitive indicators of vitamin B-6 intake than was "Towtll.

Aminotransferases as Indicator of Vitamin 3-6 Status

in this study, ALI' measurements were more sensitive to vi taniinB-6 intake than AST parameters. This observation has been dorumentedfor the plasma enzyme for both rats (2) and humanis (19). Iin addition,rat All appears to be more responsive than AST to ather physi 1ogic.st resses" (20,21). Neither AST nor ALT in plasma is con.;id, ered useful

12

in assessing vitamin B-6 nutriture in the human because of the widerange of activity in normal individuals (3,19). In experimental ani-mals, however, plasma ALT appears to be an excellent parameter.Furthermore, in our study, both -rythrocyte aminotransferases werehighly sensitive to vitamin B-6 intake.

Of the enzymatic paramaters employed, plasma AST was the leastsensitive to vitamin B-6 intake. After the first week of repletion,there was no consistent difference between the high and low vitamin in-take groups in either plasma AST or its activation coefficient. incontrast, both erythrocyte AST and its activation coefficient did dis-tinguish between the vitamin levels on all sample days in both sexes(with one exception). However, from the second week on, all repletiongroups had returned to, or surpassed, the levels of the groups whichhad never been deficient (Figures 9 and 10). Thus, the significant ef-fect of vitamin intake from day 14 on was largely due to the fact thatthe high vitamin groups had exceeded the enzymatic activity levels ofthe controls (which had been maintained on an intake of 12 mg/kg allalong). The cause and significance of this overshoot are not known.

Plasma and erythrocyte ALT were the most sensitive parameters inour study. In the deficient animals, ALT was more markedly depressedthan AST in both plasma and erythrocytes. During repletion, not onlywas the effect of vitamin intake obvious on all sample days in bothsexes, but the magnitude of the differences was greater than was foundin the AST measurements.

The overshoot phenomenon occurred with erythrocyte AST and toan even greater degree with plasma ALT. This was particularly strik-ing among all high vitamin groups, both male and female, for the firsttwo weeks (Figures 17 and 18). By day 28, these group means werenearly back to the levels of non-deficient controls. In contrast tothe high vitamin groups, all marginal repletion groups showed a largeincrease in plasma ALT only during the first week and changed littlethereafter. By the fourth week, the marginal groups still had lowerALT activity than the high vitamin groups and the non-deficientcontrols.

Plasma ALT activity coefficients of male high vitamin groups alsotended to be less than the non-deficient levels, especially during thefirst 2 weeks. Thus the overshoot phenomenon was observed, not onlyin terms of absolute enzymatic activity, but also in the relative satu-ration of apoenzyme with cofactor. Activity coefficients did not over-shoot in the females. This may be related to the fact that in thenon-deficient animals, the apoenzyme appears to be more saturated withcofactor in the females than in males (Table 14).

Erythrocyte ALT not only did not overshoot non-deficient levels,

but it was the only activity not to be restored within the 4-weekrepletion period. On day 28, the high vitamin groups were nearly back

13LJ

'o tI henon-do i Ci n t I C\' I S , b)U t LtuIeL ma ri na I vI tain ii rou;)s li-i, rc-c-OVtee only apjproxhiiite Iv ha11 t -;ay Lilumi l, teS0. r rl:u illativity wias due iin part to the iime requ re.d for- th0 dkl td cii lb 'l CII

to he rep lae'd (uiing rcd k L I I turn''ver withI er\t liroc'.'t -) n ta i ni ng_

highI)r .\IJ !ie4e

I. rvthrncvt . AlFAI i i t\,' t C~i in%,Sli ti~ \~snt i [ll,5iV i i W

d i ia tOrt ' V i taInIi 1n t-- 6 inf1talke!. ),Il hughl J IM] '; Si S fi va I l :1 te 'i~aio

S i gnli IiCleat V itaml~lin1 ii'I i k, efftc t n o u Ott )"f (i)b6 he rv, i on d11 tile-

17 Ign itI kiid C 0' th L d c i t I C I- ne e'S was; sia I I co)iimred to ot her pa ramiiit crs

v it ari i 13-1) Assayv

: tlV I lie, trganl i :,Il k IChoic Lk-Vs Lti vi Lam n 1-b-d upendcin L yeast

>i.iceno e ae s iva rum, *vIii C11 rQSpo~Tds equa I Iv t.o py r A doxi Ino,, pv ni do xa; I

am,' pv r ido ,a0in wi uder ideal r nd it: 1ins (8) . nde r l ess than i deca . cOn-

d i t l oont, hoWever , theca tsns W, I I not necessari .I N- respond equal I N' to

a11 1 1 1 ofmis o t \v i t;111 ami B1- 6 or the i-r mixtulres - h asw AsayI litd ilim i s

Lu rmu It ed to conL ini adeqitate amo unts of ll nutrients; So thajt erL'wt i

response to tile test s~ubstance i., due solely to the vitamini 1-o COntent.If thle me1d tum is not comip lute in allI other respects, two problemns may

occur: a) 1,11!;c highI vailues for Lest. sanmples and b) '"upward driftc

i.e. , nionIinca ritv (if te responlse to) test samtpl es w ih t respet' tO tlle-

Standard cuirve . 1^e experienced both Of thle a hove pro hlillt3.

A comimerc il medium (Pyridoxi no Y medium, hi feo Labo rater i es,

Ietto it, MI) wais used for thie first assays including, tile eStimatcs oif

the Vitamin conitents of tlie test meats (Table 3). Upwaird dr ift was are0curring p rob] em and was as miuchI as 200/., withI a 4-fold d if Ce renle inl

di lutionl. BUcaulse Of the drift problem and daV- to-day Variabi lity , ourConfidence in thet test mleat vi tamila vii laos Icac wOC ueb to 1e dies ired.

We next p repa redcc thle medijum reizoimiiiended by SauberlIi cl (8) and found

hat i t prODO Led greater growthI than did thle Difceo medium. Upwa rd driift

wats redLUCed hut !,is nlot c1Ti ni noted . Lif t was further dcea-,,sedt bY

add it ional supp~c~~lemn t ion~ Of thl medium Wit tlIys ilie, leic , and miethi -onile . AlI I rema' in Ii uig aIs sa s I L" s) were done W ithil thi 1S upp I ciwch tedfied i (finl.

Accordio1 ,, to Lite nierohio isil assay, tHie margiaI diet based oin

frozen eliceken Conutai ned approxiniateLy 3T. more- vi tain B-6 thian o0t(2r

meiit.-conta in i u diL- lets l I 1) . II is occeurred desp ito the faIct thatl

;11 di (h:t> had beeooreull formukled to conta in ideni]-i imiotnts of

Vi tamin Bil-0. A pk)55 i Ol c.x; liaLtion for thle U iSCi-epai;lCy hi; AS fii lows:most (94/7) oif Lte Biai t-6 in thet. frozen chicekeii diet: orijginated in

the cli lK Ien . Most Of thlis ViL t am in 1-6 was prob~alIy p rotein-b)ound (e.g.,

pyr ido)x il phtosphia ) . The I urm of Lit, vitamin inl teC otherl three hi etowas 22 t 45Z c rysta lino pyr idox inc . FreQe py idoxll icwool U be e'xpe0cted

Lo be more labile than thle protel u-bound forms. Some degraItkdat!l io Ii kelyoccurred during tife rout inc mixing, of thle mioist test metats4 With dry)

pre-mixes (exposure to moisture, light, and iir at room temperature).

Greater destruction of pyridoxine in the thermal, gamma, and electron

diets could have accounted for the decreased vitamin B-6 content in

these diets. Assayable vitamin B-6 was similar in all high vitaminchicken diets. This is not surprising because all contained relative

excesses of added pyridoxine.

Effects of Diet on Enzymatic Parameters

Brin and co-workers have reported that "rats fed X-irradiat,-d pork

were subject to marginal deficiency of pyridoxine" (22). Theirs wais

not a depletion/repletion study, but instead a simple feeding study in

which rats were fed (frozen) control pork with and without pyridoxinu

supplementation and X-irradiated pork (35% dry weight). After 12 weeks,

serum ALT was highest in the group fed supplemented pork, significantly

lower in the unsupplemented control group, and lower yet in the unsop-

plemented irradiated group. (There was no irradiated + vitamin B-6

group.) The results were completely consistent with the assavabe vita-

min B-6 contents of the meats. Thus, the "marginal deficiency of pyri-

doxinc" would be detected only when the diets are formulated to con-

tain no additional source of the vitamin.

In the present study, there was no one diet group which was dra-

matically and consistently different from all others in terms of en-

zymatic responses to repletion. There were however, trends; e.g.,

among the marginal vitamin groups in general, the group fed frozen

chicken responded the best, and those fed the semi-purified diet the

poorest. The lower responses during repletion with the semi-purified

diet can be explained by the lower food consumption. At equivalent in-

takes, this diet should have promoted responses equivalent to the, meat-

containing diets. Without a paired feeding study, however, it is

difficult to compare responses on the semi-purified diet to the chicken

diets. The chicken-based diets were consumed at approximately equal

amounts and growth rates were essentially identical for all of them.

The frozen control group usually had the highest enzymatic activ-

ities and lowest activity coefficients among the marginal groups. This

is consistent with the slightly higher vitamin B-6 content of the frozen

(marginal) diet, which was discussed above. The marginal vitamin groups

fed thermally processed and electron irradiated chicken fared almost as

well as the control group (frozen chicken) and were statistically dif-

ferent in only a few cases. The group fed gamma irradiated chicken

tended to respond the slowest of the meat groups.

The enzyme data could support a conclusion that the diets contain-

ing gamma irradiated chicken promote slower repletion of vitamin B-6-

deficient rats than diets containing frozen chicken. Differences

between these groups, especially in plasma and erythrocyte ALT activi-

ties, were statistically significant a sufficient number of times to

allow such a generalization If other factors were not considered.

15

i o..

H~owever, We hisi t (0 coll-, t t hvit gmi i rrad ii ('LI (il c uii itskean antivitamin B-0 tactoi 'r) , or if s;uch ai fact-or dole e xi!; ,I tha1t it

iSJ 1) hi 01Og i ca11llI, haad . ( )Lr reL'a3sons1 forI skept L i s-11 :1 rk

* 1 he , , J Ic I i Mcn t I I .o- I-ci! Lc e hi.1t ! c !kk h111 1 1 1 'di,'l 't Ollt lill idk-ult, i Ca Iot~ o levi 01 1 V i t J III i

Ti-b o a cc illp ka th 1) , i t w\.'lit L-e r i~Si it

test mea t in adv~inc- -Iand to s;ipp I --men eLc kih di e t 'I, cord-ing I y. Uno Ittn te ClV, the beit ava iIil, i e is sa;v for i I(total Comipos-I t e tt Vi :iui ii ls-- ?, i n- ,it u ralI M t. L'er iaIs , Lic 11

;is mea t , l it i L i z es a \ i o1~ ir)i ii i sm . I'M V L' ml 1)o Ul

which is antagonuist ic toward viE Limiri B-6 ili rai-s mighAt

just asE well bhaveI't !;iTAl 1.1rl1 ill yke !'t . I f t !I5 15 thseI

C;ISc-, tile stUd'; is; Se t-'d--i eat i Ili' We CA11110t be S111r1

whether the ILower %,avb Vi tL;! nl L- 0, inl i rrad i attcO(

CWlke n %,,;s due to des truc t ion of the v it-mn pI ) rod nt I Onof in antigonist, or both. It i S :1lafo l~Isp LOitt ic Cl IV jWc;-s ihe1 , (but no t e-al t ested ) tht gaia m i iIdit iln con-verts thle v i tam in te a ferm Iess ivai lahi e in vte rat hut

ttilit i I i zed by S. livirumi. 1We feel these, consi dcrI t'repiresent seous11 tlaws in the' o1iginail pro tocol.

*The magn it ude of the enzyime effitts and tlie end i-

tLions for detecting thecm suggest that: an antivi t 1111 )L-0fac tor (if presentL) is not biologically important. incabsolute differences between gamma iiradiated and controlgroups were, small when compared to the magnitude of t ii:changes in each group between days 0 and 7. Among1i: the

high vi tamnin groups , few diet ofCUr FeswerQ ryhSerVc' 'Iliadthey did not fit a pat tern. Thus, the del.tave2! responses

to repl et ion in the animals fed gamina irradiated chickenwere dectectable on ly under condi tions of iirgi n;i Ivi cmlillhi-h intaike and Were is ly -veryciine by add it ioma f i,,etir-v

p" 1ict(llk 11

If gamnma i rvidI atcd cli ikeii does CMIii 1 aInt \'i talil n i-I b compiounld.it shoiilId not be of great conseque.nce under normal ci rc (11(5t aices bc-cause it is a min or climpoilen t and other cons titti lin t ol thle diet we ohdcont r ibiuto enough) vitamin B-6 to compensate for the dec rca sed vitailinin the chicken.

CONC 1ItiS I (INS

OVitimin 11-6 def ici ent rciis Were- repi eted withI cl j11Ie

dI iets or d jets con ta-in1ing cli Lcken ( I rozeli , t henn-va I I v po-ud illi

or elect ron irradiated) . No dif1 e reilce Was fOundI ill irotI reSponSe

(weightL gain) among the chicken1-it'd groups . lhe gre rps- I 'd semi -

purif id d lets responded s lower, p resunia b l h0c1f c oI I wl- 100d Con-sump t i onl.

Ib

0 ALT (both in plasma and erythrocytes) was a better indicatorof vitamin B-6 status than AST under the conditions of the present

study. The magnitude of the differences between deficient and controlvalues was greater for ALT than AST. ALT parameters were not onlyslower to recover, but their recovery rates were more dependent uponvitamin B-6 intake than were the corresponding AST parameters.

0 The enzyme responses of rats fed frozen, thermally processed,and electron irradiated chicken were similar. Responses of some ofthe enzymatic paramaters were slightly delayed in groups fed gammairradiated chicken at the marginal vitamin B-6 level. No differenceswere observed at the high vitamin level. Similar results were obtainedwith both male and female rats.

0 No evidence was found for antivitamin properties in electronirradiated chicken. The amount of antivitamin B-6 activity in gammairradiated chicken (if indeed present) is minimal and probably not im-portant enough to offset advantages of food preservation by irradia-

tion.

RECOMMENDATIONS

None

17

REFERENCES

1. MCGOWN, F.L., C.M. LEWIS, and P.P. WARING. Investilption of Pos-sible Antithiamin Properties in Irradiation-sterilized Chicken.Institute Report No. 72. Presidio of San Francisco, CA:Letterman Army Institute of Research, August 1979

2. m9IN, 'l., M1. TAI, A.S. OSTASIEVPR, and H. KALINSKY. The relativeeffect of pyridoxine deficiency on two plasma transaninases inthe growing adult rat. J Nutr 71:416-420, 1960

:i. SAUBERLICH, II.F., R.P. DOWDY, and I1.11. SKALA. Laboratory Testsfor the Assessment of Nutritional Status. Cleveland: CRC Press,Inc., 1974

4. SIANE, 11. Vitamin B-6 and blood. In: luman Vitamin H-6 "qequire-ments. l'ashington, DC: National Academy of Sciences, 1o78.pp 111-128

5. TIUIELF, V.F. and H. BRIN. Availability of vitamin B-r vitarersfed orally to Long-Evans rats as determined by tissue transani-nase activity and vitamin B-6 assay. J Nutr 9,1:277-242, 1969

6. ,EA']ON, (;.*. and MI. CIIENEY. Vitamin R-6 requirement of the rm:lealbino rat. J Nutr 87:125-132, 1965

7. ,OGERTS, Q.P. and A.E. HAPPER. Amino acid dlets and maximal grow:thin the rat. J Nutr 87:267-273, 1965

S. SABIMLICIH, 1;.E. Vitamin g-6. In: The Vitarins, Volume VII,edited by P. Gyorgy and W.,. Pearson, New Yor!:: Academic Press,19 17. pp 169-208

9. INTERNATIONAL, Fr1ERATTON OF CLINICAL CFIFT'1 TRY. Provisional re-commendations on JFCC methods for the measurement of catalyticconcentrations of enzymes, Part 3. Revised IFCC method for as-par'tate arinotransferase. Clin Chem 24:720-721, 197S

10. TlL COT IIl'E ON N"ZYMES 0! TlE SCANDANAVIAN SOCIETY FOR CLINICALCIIISTRY AMD CLINICAL PHYSIOLOGY. Recommended methods for de-terminations of four enzymes in blood. Scand . Clin Lab Invest33:291, 1974

11. FlRALA, J.1., P.P. WARNING, M.F. LYONS, M.G. t:USNAK, and J.S.ALLETTO. Methodology for determination of blood aminotransferases.In: ,lethods of Vitamin P,-6 Nutrition, edited by J. Leklem andR.D. Reynolds. New York: Plenum Press, 1981. pp 171-202

18

12. TECIINICON INSTRUMENT CORPORATION. Technicon Method No. Sr4-0010FH4. Tarrytown, NY, 1974

13. KESSLER, G., 1R. RUSH, L. LEON, A. DELEA, and It. C11POI.A. Atuto-mated 340 nm measurement of GOT, GPT, and LDI!. Volume I. In:Advances in Automated Analysis, Technicon International Congress.Miami, Florida: Thurman Associates (1971), 1970. pp 67-74

14. BERGMEYER, H.U., P. SCIEIBE, and A.W. WAIILEFELO. Optimization ofmethods for aspartate aminotransferase and alanine aminotransfer.ase. Clin Chem 24:58-73, 1978

15. SIGMA CHEM2ICAL COMPANY. Technical Bulletin No. "7-1WV (7-79).St. Louis, MO: Sigma Chemical Conpany, 1979

16. UNIVERSITY OF CALIFORNIA. Biomedical Conputer Programs. LosAngeles, CA: University of California Press, 1975

17. I'INER, B.J. Statistical Principles in Experimental Design. NewYork: McGraw Hill Book Co., 1971

18. ItOLLANDE,, M. and D.A. IWOLFE. Nonparametric Statistical Methods.New York: John Wiley & Sons, 1973

19, BAYSAL, A., B.A. JOHNSON, and t1. LINKSWILLER. Vitamin B-6 deple-tion in man: Blood vitamin B-6, plasma pyridoxal-phosphate, serumcholesterol, serum transaminases, and urinary vitamin 11-6 and 4-pyridoxic acid. J Nutr 89:19-23, 1966

20. BRIN, M. and R.W. MCKEE. Effects of x-irradiation, nitrogenmustard, fasting, cortisone, and adrenalectomy on transarinaseactivity in the rat. Arch Biochem Biophys 61:384-389, 1056

21. AWAPARA, J. Effect of protein depletion on the transaminatingactivities of some rat organs. J Biol Chem 200:537-541, 1953

22. BRIN, M., A.S. OSTAS|EVER, H. TAI, and I. KVALINSKY. Effects offeeding x-irradiated pork to rats on their pyridoxine nutritionas reflected in the activity of plasma transaminases. .1 Nutr75:35-38, 1961

19

LIST OF FIGURES

Page

Figure 1 Growth Responses of Vitamin B-6 DeficientFemale Rats Repleted at Three Levels ofDietary Pyridoxine (Pilot Study) ... .......... 23

Figure 2 Erythrocyte Aspartate AminotransferaseResponses of Vitamin B-6-Deficient FemaleRats Repleted One Week at Three Levels ofDietary Pyridoxine ...... ................. ... 24

Figure 3 Growth Curves, Phases 1 and 2 ...... ............ 25

Figure 4 Growth Curves, Phase 3, Groups C and D ......... ... 26

Figure 5 Growth Curves, Phase 3, Groups E and F ......... ... 27

Figure 6 Growth Curves, Phase 3, Groups G and H ......... .. 28

Figure 7 Growth Curves, Phase 3, Groups I and J ... ....... 29

Figure 8 Growth Curves, Phase 3, Groups K and L ......... ... 30

Figure 9 Erythrocyte Aspartate AminotransferaseActivity, Males ....... ................... ... 31

Figure 10 Erythrocyte Aspartate AminotransferaseActivity, Females ...... .................. ... 32

Figure 11 Erythrocyte Aspartate AminotransferaseActivity Coefficients, Males .... ............ ... 33

Figure 12 Erythrocyte Aspartate AminotransferaseActivity Coefficients, Females ..... ........... 34

Figure 13 Plasma Aspartate Aminotransferase Activ-ity, Males .......... ..................... 35

Figure 14 Plasma Aspartate Aminotransferase Activ-ity, Females ......... .................... 36

Figure 15 Plasma Aspartate Aminotransferase Activ-ity Coefficients, Males .... ............... .... 37

Figure 16 Plasma Aspartate Aminotransferase Activ-ity Coefficients, Females ..... .............. ... 38

APPENDIX A

21

P1ECZDIM PAtZ UJJ-MT 721j=

Page

Figure 17 Plasma Alanine Aminotransferase Activ-[ty, Males ................................. 39

Figure 18 Plasma Alanine Aminotransferase Activ-ity, Females ........ ..................... . 40

Figure 19 Plasma Alanine Aminotransferase Activ-ity, Males ......... ....................... 41

Figure 20 Plasma Alanine Aminotransferase Activ-ity, Females ........ ..................... . 42

Figure 21 Erythrocyte Alanine AminotransferaseActivity, Males ......... ................... 4

Figure 22 Erythrocyte Alanine AminotransferaseActivity, Females ........................... 44

22

p4-)

44

4c N

4-

L) 4-)

4-

4 -o1)

M

a)W

Ll 4-

0232

2M

AN

18A

CT

V

C0 14E 4FF

C

I 22

MG DYRID0xlINE/'KG DIET

70

A 60

A

0 0

u 30-

T21

0 2 4 6 a 0o 12

Mr, DYRD)XINE.K'G DIET

Figure Z'. Erythrocyte aspartate aiinotransferase responsesof vitamin B-6-deficient rats (females) repletedone week at 3 levels of dietary pyridoxine. Uppergraph, activity coefficient, lower graph, enzymaticactivity in (arbitrary) chart units. (Pilot ,tudy).Vertical bars represent mean SD

2 4

EA In6NR lee

AT 140

£ 12°I Ise

HT

RAM 40S

23

A -A NON-DEFIMIT8 , - DUIMENT

135 ?9 It13 57 18 2123 2S27 2 1 33 35DAYS

408.

E 340AN

SMR 280AT 20

2410V1E 220

1 2w

InT In* 141

* 120

A asH

as4

41 .A - NMN-Ccx2.i - DEIFrX

0 2 4 6 8 181214aIlS623224232S39S2343,3S4424446

DAYS

Figure 3. Growth curves, phases 1 and 2. Females (upper), males(lower).

25

E 269AN

R 240A

W 220E

H

IS

A

1 Ja A -- NON-DEFCZNT

140 :C -- SEMIPURIrIED £HAR3INALI140:D -- SEIIPURIF-IED CHIG3

O 2 4 8 89 10 12 14 16 IS 20 22 24 2S 28 30

DAYS

400

M 440EA 420N

460R

360

E 340

H 320..

280 --G -R 260

M 240

290:D -- SEMIPURIFIED CHIGH3

0 2 4 5 8 10 12 14 16 18 20 22 24 2e 28 30

Figure 4. Growth curves, phase 3, Groups C and D. Females (upper), males(lower).

26

268

E 2M0AN

R 240AT

4 220--EI M.

s 292 /'-.

T "/G 188RAmS 16

A-- NON-DEFICIENT140 ... E --FROZEN CMARGINALU

-4--:F-- FROZEN CHIGH)

0 2 4 8 8 10 12 14 le IS 20 22 24 26 25 3U

DAYS

468

H 440EA 420

408RA 38

348

H 328T

/ 2829R 26 .AH 248S

228---" :A - NON-DEFICIENT

29 . :E -- FROZEN (HARGINALD:F - FROZEN CIHIGH)

0 2 4 8 8 10 12 14 16 18 20 22 24 2e 28 30

DAYS

Figure 5. Growth curves, phase 3, Groups E and F. Females (upper). In,1les(lower).

27

260

E 2MAN

R 240AT

W 220E7G 2eeH/T

G 180RA

A - NON-DEF71CIENT---------------- G - THERMAL CIIARGINAL)

aM - THERMAL CHIGH)

0 2 4 e 8 10e 12 14 16 28 20 22 24 20 28 30

DAYS

EA 420N

400RA 388 . 5

340

E 34

G 320H 300T

/ 280GR 2680 -

M 240S

220 - :A -NON-DEFICIENT

20G- THERMAL CMARGINAL)ii -iH -- THERMAL CHIGH)

0 2 4 e e 10 12 14 16 18 20 22 24 2e 28 3e

DAYS

Figure 6. Growth curves, phase 3, Groups G and H. Females (upper), males(lower).

28

288

ME 20AN

R 240AT

E

I /.G 200HT

S lee/ /

:A -- NON-CEFICIENT140. -- GAMMA CMARGINAL)

--'-- :J -- GAMMA (HIGH)"-I 1 -T- I I I -T

0 2 4 6 8 18 12 14 16 18 28 22 24 26 28 30

DAYS

460

440EA 420N

4e0

A 380

T 360 o

4 -----

W 340

G 320

H 380

/ 280S

R 2608AH 240

: A -- NON-DEriCIENT200. :1 GAMMA CMARGINAL)

:-- GAMMA CHIGH)

0 2 4 6 0 10 12 14 18 18 28 22 24 2e 28 38

DAYS

Figure 7. Growth curves, phase 3, Groups I and J. Females (upper), males(lower).

29

280

E 260A -

N

R 2-8 0A -

T $

W 220

E

H/T 1

G ISORA

S 16e 1.A tON-DEFICIENT

140 - i - ELECTRON (MARGINAL)I '~:L -- ELECTRCN CHI3i

I I I I I I I I r- -7'** - 1 **-i----*I T-------*---r-*-*-*-I-*0 2 4 6 8 10 12 14 16 18 29 22 24 26 28 30

DAYS

468

M 440EA 420N

A 38T 360

340E

T280

G -

R 288A 7H 2408S -'

220 --------------- :A -- NON-DEF7ICIENT208 -~ -- 1- i ELECTRON (JIARGINAL)

:L - ELECTRON CHIGH)

0 2 4 8 8 30 12 14 18 I8 20 22 24 2e 28 30

DAYS

Fiqure 8. Growth curves, phase 3, Groups K and L. Females (upper), males(lower).

3u

2.4

2.2

2M

N 1.4

I P.4

U""2 Day 7H ~~1.2 U..4

L I .r

0.6C*

0.4

0.2h-'HIGH*--CMARGINAL

D0EF NN-Oc..r s$?-P MrOZEN THERMAL GAtMA ELECTRON

2.4 I

H 1E 1.6 8r '-.AN 1.6

I 1.4U 1 Day 14/ 1.2

L

R1 3 .SC

0.6

0.4

0.2

S---VMARGINALI I I ' I

ocr NCN-Ocf SEM1H-P FRO'ZEN THER"MAL GAfiMA ELECTRCN

2,4

2.2

S.. ... .. .

A

"I 1.4UU 1.2 Day 28

ML

C

.6

I IG

DEF NON-DEF SEMIi-P FROZtN THERMAL GAMMA ELECTCON

Figure 9. Erythrocyte aspartate aminotransferase activity, Id h.(Group means) Symbol at far left representsdeficient group at Day 0.

31

I Day 7

L

* .aC

9.6

8.4

3.2 b***4 4H

M7 MN-OL'W 9341-0 FROM4 1gThEML a.EC7XCN

2.4

N 1.6

U Day 14

3.2

2.2 .

S 1.2

L

C

Figure 10. Erythrocyte aspartate aminotransferase activity,females. Symbol at tar left represents deficientgroup at Day 0.

32

A C. ---4 MARINALN

A 1.CTI 1.7VIT .Y Day 7

1.5C0 CE 1.4

F 1.3 "". ..... . ..p-.ICI 1.2

NT 2.

I I i I i

DIF NON-DC? SEMI-P FROZEN THERMAL GAMMA ELECT"CN

M 24E &- HIGHA 9 --- MARGINALN

A 1.0CT1 1.7V

T 1.6y Day 14

f.5C0E 1.4FF1 .3

C .1 1.2..

-...

N 3

A 1.8

i , I I

D£? NON-OCT SENMI-p TROZE4 THERMAL GAMMA E"LECTRCN

N 2

A 2--91A61AN 1.7

A 15]

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T .6Y .Day

28C0

E 1.4F

F 1.3ICI1 .2E o ..... .. -........ ........ .......T 1.l1'

I I , I I

DIF NON-OCT SEMI-P FROZEN THERMAL GAMMA ELECTRCN

Figure 11. Erythrocyte aspartate aminotransferase activitycoefficients, males. Symbol at far left representsdeficient group at Ddy 0.

33

M 2r

-Yl

E - H I G HA 19 r-- MARGINAL

A 1.

CT

'V

T 1.6

TY I s Day 7C0E 1.4F.

1.3 1L.C

2 "'11-°°° -... ........

T l

f I -- t I "1 I I

DE" NON-DEF SEM-P FROZEN THERMAL GAMMA ELECTRON

M 2E 1H IG 1-HA,, •

@ --- 40 M S.j

A .8C1TT 17

V

YT -6

C .: Day 14

CF

C1 1.2

E ..............

1 I I I I I

DEF NON-OEF SEMI-P FROZEN THERMAL GAMMA ELECTZON

E h HIGHA 3 --- QmAR6NAL

N

A i.8

T1.7

1.6

1 Day 28I.SC

1.4

F

1.3C7 1.2

"I,, o " ..... ' "- ....... ......

I I I I

DE" NON-DEF SEMI-P FROZEN THERMAL GAMMA ELECTRON

Figure 12. Erythrocyte aspartate aminotransferase activitycoefficients, females. Symbol at far left repre-sents deficient group at Day 0.

34

4S

48

A .N 36 I

8I Day7/

L 26IT 29rR

IS

H- IGH

s

* ± --4D IARGNALiI i I ' I 5

D£7F NON I-OcF S£MT.-P rRoz..rN TH"£RN'AL GAIINA £L£FCTRON

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30

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Is

t

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L. 26

29E

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DEF NON-DOC SEMI-P FROZEN THIERMAL GAMMA ELEC7RON

Figure 13. Plasma aspartate aminotransferase activity, males.Symbol at far left represents deficient group at

Day 0.3S

S

5

Ms

EAN 3S ---- -- .........

1 30u Day 7L 2SIj 20R

is

HIGH0-1MARGINAL

I I | I 1 I 1

DrF NON-DEF SEMI-P FROZEN THERMAL GAMMA ELECTRON

SS

45 0

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30 Day 14L 2S

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L 302

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R 20

IB4

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Figure 14. Plasma aspartate aminotransferase activity, females.Symbol at far left represents deficient group atDay 0.

3b

I.. , .

2.6 4E 2.5 * HZGHA 0- MARZNALN 2.4

A 2.3C 2.2T: 2.1V

T 1.9V 1.9 Day 7Y

1.'

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" ' *"

N 1.2

TI.'

I I!! i

O" NON-DEF SEN-P FROZEN THERMAL GAMMA ELECTRON

M 2.:

S 2.-- HIGHA 4

MAR INAL

A 2.3C 2.2

1 2.!yV 2_

T 1.9 jY . Day 14

C 1.7

E 1.6

F I.S1 1 .4"" ' "IC is3 ......... . ... ""

N 1.2T

0CF"

NCN-OC SrfMI-P FROZE.N THERMAL CAM"MA ELECTRON

M 2.8

E 2.5 * ' HIGHA -- 0 MARGINALN 2.4

A 2.3

C 2.2

1I 2.1

1.9Y Day 28

C

0 1.E£ 1.6

F I.5

1. | 4 .. .. ..1 1.3 . . . .. . ,. "- -........

N 1.2T f.

DE" NON-OEF SEMI-P FROZEN THERMAL GAMMA ELECTRCN

Figure 15. Plasma aspartate aminotransferase activitycoefficients, males. Symbol at far leftrepresents deficient group at Day 0.

37

.- . . . ......... ..-. .-.

2.7c 2.0N 2.S MARGINAL

2.4AC 23T 2.2

V 2.1I

T 2Y t'9

C ,.0 Day 7a 1.7

£F I.F F 1.5IC 1.4 ..I "...E 1.3 _ _ _ _ _ _ _ _ _ _ _ _N 1.21"

1.1

DEF NON-DEF SEMI-P FROZEN TIERMAL GAMIIA EL.ECTRON

M 2.7NE 2.8 a- NIGHAN 2-5 3--QMARGINAL

2.4

AC 2.3T 2.2IV 2.1

2TY 1,9

C 1.6 Day 140E

F 180F 1.5IC 14

C 1.4 - -

I lpI I....

0E" NON- EF SEMI-P FROZEN THERMAL GAMA ELECTRON

M 2.7

E 2' a- HIGHN 2.5 1---C MARGINAL

24

C 2.31T 22

I

Y f a

c t.6 Day 280 1.7EF I.O2F .

C .4E .3 . ... "

I T .21T

.I1 T 1 I I

DEr

NON--EF SEMI-P FROZEN THERMAL GAMA ELECTRON

Figure 16. Plasma aspartate aminotransferase activitycoefficients, females. Symbol at far leftrepresents deficient group at Day 0.

38

38

14 26EAN 20

I

u " ---------.k. Day 7L 1

Or

R Is

"-''mARGNAL

I I I I

D0E NON-DEF SEMI-P FR07EN THERMAL GAMMA ELE CTRCN

38

2S

M

AN 29

1 a Day 14.°U ... .. Da

L o r -ITER lB

S

HIGH0---0MARGINAL

I ..I l I I I I

DF NON-DEF SEM -P F1O2EN THERMAL GAMMA ELECTRON

38

25E

AN e

1 0u Day 28LL -*." .

ITER IS

SHIGH

G,-4D MARGINAL

I I - - ' I ' I

OEC NON-OET SEMI-P rROzEN THERMAL GAMMA E.ECTRON

Figure 17. Plasma alanine aminotransferase activity,males. Symbol at far left representsdeficient group at Day 0.

39

- __ ____ ____ ____ ___

30

E 2A0

L IEL is .1

DEF NON-OEF SEMI-P FROZEN THERMAL GAMM ELECTRO

30

25.

2. --------...- .........

Day 14

E

-MARGINALSI I

OEF NON-DEF SEMI-:' O7EI TFRROE AL GAMMA ELECTRON

3 i

25

A -- - - - .. .. . ..

u" .......... Day 28

L 'S

R IQ

Si

gi"( MARGINAL

DEF NON-OEF SEMI-P FOZEN HERMAL GAMMA ELECTRON

Figure 18. Plasma alanine aminotransferase activity,females. Symbol at far left representsdeficient group at Day 0.

40

ME 2 *.HIGHA 1--- MARGINALN L

AC 1.8TIV 1.7

IT 1.8IS .Day7

C .

F

C -5", '° ".

I 1.3C

NT 1.1

p I I

DEF NON-DEF SEMI-P FROZEN THERMAL GAMMA ELECTRON

M

A HIGH

N 3---E MARGINAL

1.9AC 1.8T

V .

TT I.e *JY Day 14C 1.50E ------.. ...-- . .. ...-

F 1.4 --- aF1 1.3CE f.2 ""0/

ENT t.I

I I I I

DEF NON-DEF SEMI-P FROZEN THERMAL GAMMA ELECTRON

E 2 * HIGHA a --- OMARGINALN 1.9

AC 1.8

TIV 1.7

T 1.

C .Day 28C 1..a . . -. ... -... ,

TI I .3:-"

.

I i32 0",

DEF NON-DEF SEMI-P FROZEN THERMAL 1AIIMA ELECTRON

Figure 19. Plasma alanine aminotransferase activitycoefficients, males. Symbol at far leftrepresents deficient group at Day 0.

41

M 2.sE 24 *HIGH

A M ---* MARGINALN 2.3

£ 2.2CT 2,1

V1TY tDay

7C t.70E t.F t.SF1 1.4C1 1 3 0- - -.. .............

N 1,2

E I .I

Er

NON-CDE SEMI-P FROZEN TMiER.MAL GAMMA ELECTRON

M 2.5

E 2.4 & HIGHA - MARGINALN 2.3

A 2.2CT 2.1 -

I 2

Y .8 Day 14C 1.70 1

F -F 4

2.S .4CI 1.3 Day 2S 1.2.

00

i I I T

DEF NCN-OEF SEMI-P FROlEN fl'ERlAL GAIIMA .LECTR1ZN

2.5 .4

E

1- 2.4 . 'sHTG

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N 2.3 "

N 2.2 -a

|r

T 2. -

.ns dg Day 28

0

F .5

S .4 1E >1N 1.2.o......, .- . ......... .......

I I r I

01W"F NOIN-.0F SEMIT-P IrFPZE'N THE'RMAL. GAMMA ELECTRON

Figure 20. Plasma alanine aminotransferase activitycoefficients, females. Symbol at far leftrepresents deficient group at Day 0.

42

0.20

*---^MARGINAL0.26 3 '--- 1tlIG11

0.24

M 8.22

E 02AN e.19

X 0.18U/ 0.14 Day 7L 0.12 3"

A /~R

C .8

.84

*

.8_2

DEF NO-DEF SEMI-P FROZEN I1FRMAL GAMMA ELECTOON

e.280 bMARGINAL

0.26 - HIG11

0.24

N 8.22 ---------

E .2 " . .--------- ------

AN 8.18

Day 14

L 9.12

0.1

C *

.66e

.04

.82

I I IF VI

0EF NON-DEl SMl.-P FROZEN THERMAL GAMMA ELECTRON

8.4 MARGINAL8.32 - 51H16H

0.3

N 08.28 0 ....... .......... -,.

EA 8.24 "0r

N .22

I 8.2u, 8. e Day 28C 8.16

9.14

0.12C O.1

.084

.028

DEF NOWi-CEF SEMI-P FROZEN THERMIAL GAMMA ELECTRON

Figure 21. Erythrocyte alanine aminotransferase activity,

males. 43

.

8.34 M MARGINAL8 3? " "'rHIGlI

0.3

9.28E" 9 26

A 9 24N

0 2?1 92U/ 0.18

0 15 Day7L6, 4 ..8-..

C 8.I

.88

.84

DEF NON-DEF SEMI-P FROZEN THERMAL GAMA ELECTRON

, 34 6-- MARGINAL

0.32

8.3

e. 28M 9.26

A 9.24N

0.22

I e.2U 1 -eDay 14

L

R 0.1284

C 9.1

.98.0

.94

.920 T-"'-I [ T |--r

DEF NON-DrF SEH]I-P FROZEN THERMAL GAMMA ELECTRON

0.34 K 'AR6INAL

9.32

0.3

6 28

0.2E i2'.' " 3......A 924

0.22

I 0.2u Day 28

0 .166.2

R8 0.12

C 8 t

08

04

62

DEF NON-DEF SEMI-P FROZEN THERMAL GAMMA ELECTRON

Figure 22. Erythrocyte alanine aminotransferase activity,females.

44

LIST OF TABLES

Table 1 Vitamin B-6 contents of chicken test meatsand repletion diets ....... .................. 47

Table 2 Schedule and diet codes for anti-vitamin B-6studies .......... ........................ .48

Table 3 Summary of quality control data for plasmaand erythrocyte aminotransferase activity ......... .. 49

Table 4 Growth of pyridoxine-deficient rats repletedwith semi-purified or chicken-based diets (males). • 50

Table 5 Growth or pyridoxine-deficient rats repletedwith semi-purified or chicken-based diets(females) ......... ....................... .51

Table 6 Analysis of variance (ANOVA) significancelevels for erythrocyte aspartate aminotrans-ferase activity ........ .................... 52

Table 7 Summary of erythrocyte aspartate aminotrans-ferase activity in pyridoxine-deficient ratsduring repletion with semi-purified chicken-based diets .......... ...................... 53

Table 8 Summary of erythrocyte aspartate aminotrans-ferase activity coefficients in pyridoxine-deficient rats during repletion with semi-puri-fied or chicken-based diets. . . ........... 54

Table 9 Analysis of variance significance levels forplasma aspartate aminotransferase activity ........ .55

Table 10 Summary or plasma aspartate aminotransferaseactivity in pyridoxine-deficient rats duringrepletion with semi-purified or chicken-baseddiets . . . . . . . . . . . . . . . . . . . . . . . . . 50

Table 11 Summary of plasma aspartate aminotransferaseactivity coefficients in pyridoxine-deficientrats during repletion with semi-purified orchicken-based diets ....... .................. 57

Table 12 Analysis of variance significance levels forplasma alanine aminotransferase activity ........ .. 58

APPENDIX B

45

Page

Table 13 Summary of plasma alanine aminotransferaseactivity in pyridoxine-deficient rats dur-ing repletion with semi-purified or chicken-based diets ........ ..................... ... 59

Table 14 Summary of plasma alanine aminotransferaseactivity coefficients in pyridoxine-defici-ent rats during repletion with semi-purifiedor chicken-based diets ..... ............... ... 60

Table 15 Analysis of variance significance levels forerythrocyte alanine aminotransferase activ-ity ......... ......................... .... 61

Table 16 Summary of erythrocyte alanine aminotrans-ferase activity in pyridoxine-deficient ratsduring repletion with semi-purified orchicken-based diets ........ ................. 62

Table 17 Summary or erythrocyte alanine aminotrans-ferase activity coefficients in pyridoxine-deficient rats during repletion withsemi-purified or chicken-based diets .... ........ b3

46

00N Cd

~4) . m .o C

ID- -4

CJ0 41

0- ..4 LA 0r_4 0N41 -4 V

to 00L L A) W 4.)

0 CNN

C14 . .n

+1' +1 +1

LA LA LA) 05)

C)4 -4 *

000

4.' 4) 1

11) CO 9Z -

ca 9. +1 +1 +1

0 LO '0) '0) X0Uot +.' t.l 04 00 r- tO LA L LA0

as4 CO : 3

4-)4-

4)~4 N NU*-

U) 0

4-4 '044C.14 NO 4 000

fn '-4 r4

S00 -- 10

4) Q0

0~ +j4' .0

4-))

-4- .. 4 )

co4 U 4 0 +j 4

4'4 3c 4)'-q )

.1 4-J -4 -4U)

>) 4J c: 4.1v ' tnV

4) 4J4 .14i4)C

oe ' ' t*4 bU -4 r-4 On -4 U -40- ~ ~ -4 - 0 rA >- X

4J -3 U3 4J (U It' 4 '0 * 3 ' 0 I ~4) 4).~ ~

H> > >-

47

Table 2. Schedule and diet codes for antivitamin B-6 studies1

Pha:,;e Length of Phase Diet Groups

1. (Quarantine) 1 week A

2. (Depletion) 33-42 days A,B

3. (Repletion) 4 weeks A, L-L

Vitanin B-b Level

Diet Code Diet mg/kg dry weight.

ASenipurified 12.0 (non-deficient con-trol group)

B Semipurified 0 (deficient diet)

C Semipurified 2.5

1)Semi puri fi ed 12.0

Chn i Iii in

Frozen Chicken 2.5

Frozen Chicken 12.0

G Thermally Processed 2.5

IIThermally Processed 12.0

IGamma Irradiated 2.5

.1Gamma Irradiated 12.0

Electron Irradiated 2.5

I.Electron Irradiated 12.0

'The cx(xlri menta I des ign was ident ical for both studies except one used a Imale animals and the other all females.

48

Table 3. Summary of quality control data for plasma and erythrocyte amino-transferase activities

Within Day Precision Between Day P'recision

Mean S.D. C.V. Mean S.D. C.V.

Plasma AST1

Normal 2 19.1 1.08 5.65 19.2 0.42 2.17Normal 3 23.4 1.17 5.00 23.4 0.88 3.73Elevated4 42.1 1.87 4.44 42.1 1.31 3.1]

Plasma AST StimulatedNormal 3 36.4 1.39 3.82 36.4 0.69 1.88Elevated4 68.9 2.07 3.00 69.1 1.53 1.21

Plasma ALT1

Normal 2 21.5 1.12 5.21 21.4 0.82 3.83Normal 3 30.5 1.07 3.51 30.4 1.23 4.05Elevated4 86.2 2.89 3.35 85.9 3.21 3.73

Plasma ApT StimulatedNormal 34.4 1.89 5.49 34.3 1.21 3.62Elevated4 92.5 3.64 3.93 92.3 2.65 2.87

Erythrocyte AST5

Normal, Male Rat 1.91 .050 2.62 1.90 .047 2.47Deficient, Male Rat 0.50 .028 5.60 0.49 .017 3.46Normal, Female Rat 1.86 .052 2.81 1.85 .059 3.19Deficient, Female Rat 0.76 .041 5.46 0.75 .025 3.33

Erythrocyte AST, StimulatedNormal, Male Rat 2.09 .053 2.52 2.08 .083 3.99Deficient, Male Rat 1.06 .044 4.20 1.05 .034 3.24Normal, Female Rat 2.06 .060 2.90 2.05 .097 4.73Deficient, Female Rat 1.32 .060 4.57 1.31 .071 5.42

IlU/liter. 2Dade Monitrol I; American Hospital Supply, Miami, FL. 3Sigma EnzymeControl -2N. 4Sigma Enzyme Control -2E. IU/ml packed red cells. Appropriatepools were prepared in advance, divided into aliquots and stored frozen at -700C.

49

'-41

to 00 mcOi C t) ~0 '0 --4 C'.3 00 t"

7j iz:D

:00

C~l C)C)I

U0U

0 ~ . C Ci -l ito t- :0* Cc 0) 1 - -(1 )

r --C )(:

7.04 r)'- - '. 0 . 0 1C- r0 -- '0 -

a)O Cd

5-. C

C) a

CI C) -.t 1-: (13 rI CD- C

CZ t - to r4 44 r1 to t4 to to

C )D

4 -'14 ,~C , . ,4 , Cl .444 r- o44 '4 ().4 a 4 4 to C

T) fO tc a -zt It I I ' IT j r- C '41

C*, 4 -- ' + 1 41 +1 41- +1 +1 1 4 1 +I1- +1 -f- I

I-I I-) L

4j4L

0 0-4-4 C)

z ~ c 4- '0 a) 0) a i) a a ) a

0:

C 0t

Ob

-.4

44)

L4~~~ '-4 -0 'I (0(0 00 CIO~C

4) CU0 0

CU 4

4) 0 -T t , o l 0t((4.4r ( 1 4 4 1

44 (D

4) 0

4J

00

m . . 00 -1 ZA ND fl, (O 0 ' A 00

00 00 t) 0 c

a) C

1- D4; t.0 -4- 4 - - 4 -1 -

o -

44) CCUi4.4 4)

444-

-4 4)

.00

CdC

L)A 4 ~'N 04 U oA~

Table 6. Analysis of variance (ANOVA) significance lvels for eryth-rocyte aspartate aminotransferase activity.

Erythrocyte Aspartate Aminotransferase Activity (Unstimulated)

Vitamin Diet Interaction

Males

Day 7 .000 .000 .000Day 14 .000 .139 .035Day 28 .000 .0262 .517

Females

Day 7 .000 .001 .023Day 14 .000 .080 .297Day 28 .022 .527 .282

Erythrocyte Aspartate Aminotransferase Activity Coefficient

Ma I es

Day 7 .000 .000 .000Day 14 .000 .0003 .000Day 28 .000 .000 .000

Females

Day 7 .000 .000 .000Day 14 .000 .034 .107Day 28 .000 .007 .004

'P values obtained by ANOVA on 5 food groups, 2 vitamin levels (GroupsC through L). 2Variances were significantly different by Bartlett'stest. Kruskal-Wallis nonparametric analysis revealed no significantdiet effect. 3Diet differences were not significant (P < .05) byNewman-Keuls test. Examination of 95% confidence intervals did revealapparent differences.

52

000 00 00 0. .0+...1+-) 04

d4J+1 +1 +1 +1 +1 +1 +1- +1 +1 41 + ! +1 V LI)

r-40t l 0

O"'r 0 ) 1 Ot)0 C) 0) ,

4, cl ... .

tn mtn o LOtn l) n W)10 Zil -4 .,1

lz. Cj

0 cl -1000 0 ~ o00 000 t t 000 V-H4IT

-4 tm Q Cd -4 cl'-1

-14 IHT-c Lt)-4O IT0 0otn" ) t nV

.0 r -

4J 4J *q *H ui 14H a)*- +1 +1 +1 x +1 +1 +1 X< +1 +1 +1 0 +1 +1 +1 Ln

> -4F- x 0 0 -4 ~ 4j *0-H0 T00 o " 0 &i tJ "0 'D t) C H 10 mO0 a) -4

4J V mN~O *H 1)0 0 -H TT~ s C -4 10O' m 0 )0 CD(u. 734 -r.4 -' r.

5.4 CL4.- C'JC' -1C 0. -4 V) u 0

US ..- Ef (Cd~~) N.. 4- 44

F4 = 0 00 . - 00 00-0

44 14.d 4 a0 0a)1 *H 0q t- , -t 'T - L D1 -

-4C4 ) .0 4z)z 1 0 -

C* . . . . . "0

4J+1 +1+1 +1 +1 +1 tA +1 +1+1 (n +1 +1+1 -aV* )c0 ) (n 0) 4) 4J (D - 4=0 t ) f 10 N-O) .L i ) r\ - 00 * ('I -4 (1U *-

S%. "o *4 c . . . . ~ 4 4U*H 4) 0-4 ,-4 ,-1C r~,4 -1 4-4 .- C'(N +1

.4 " -0 4 - 4

Q)0 44CI 00 0001.14 *H * C

1:0

(D 13 C 0 -

00) Q,4. +1+1+1 +1 +1+1 +1 +1 +41 +1 +1 .- 4 00 r

Y0 '0000 p.) 00.41. r-0,- 4- *0 oC a

0 30

: 5-4 4)

>. -4-4 5-4 ( 4)us4) f-'0 C) ~t0 C>(l0 r-.l-0 C) g0 4

-4J 4('4 .H ' .. 4 ..*...0 (v ('I t 1+ 1+ 1+ 4

+j Q)) a) IH Us: -o *or I -

0 0\0 0 0 t- 0% 00053

-4r-4-4 CDr4 C) -4-11- 1 "1 Q +

41) -f- +1 +1- +1 +1 +1 +1 +1 +; +1- +1- -+- t, CC: 4j -4

Xd CA-4- "1-4 -14'- -4 1-4 -4 It

-4 L Cn

-4 40 -1' -4 -4 C' Lo 4-f-'-4 tr2 000 CDC00D C 0 C0 0 000

41 - * . . . . 44 JI ) Il

4- 4+1 +1+1 +1 +1 +1 +1 +1 +1 +1 +1 +1 C) r---"In 9 -4 0

7) V)- ri- 14- -1olr- -4 tC J-4 C lto Nr 04 ' 1NJfv- 4-4 v4

.- 4 V) 14,. - - - . 4 r.4 z''4 'i *4-4 Id "7 t' C)

0-0 n4U d , 4 C-

-0 z -. 00 C)00 C)00d 0u - C 5 -4 C) C) -4 -1 -. Q -

C.),- CD -H QH 11D 0) CD () 0 0CD r

-4 +1 +1 +: x< +1 +1 +1 X< I +1- -+1 0 +1- +1 +1i ~ .> - 0 0 = Z

-1- 0 m 00 ~0 '0 L ' ~tn (NJ--- 'IT tn 0 C:) L')4 - -~(J'. - H vfvfvf -- N -4 '-4 5- - v- 4 =f . ;Z

5-4 .- ,-4 -vf-4 -1-4 -- 4- +1 U)

k)C -,q 5 )-I

0 -f-4 0: 0f 4- ~ - 04,4 0o to .d .44 0I dv

tn* 'A (NI -40'- (J -'CD -4 v-q0I fN C 000 v-4 0D0 0 0 0CD 0CD0 +1 - 4-) '

44 C) 0+1 +1+1 +41 +1 +1 (A +1 +1 +1 tn1 + +1 1i Ci .,q 440~f V ) V) V) ) a) WC) u U r0

00 U) C)aOOf ) \0 \0 I , " 0 rt' - 4 5 ~-.- 0J:0 v-4 CA 1 " -4 --- Cd r-4 4 1-4 1 -4 -4 , 44

EU4 -. CI .- f- . .4 4- C) .- ~H- . ) . ~ - d 5-

-H, 0 -1z

C0 -H-H a) r0

5-4 4-4 -H (NJ -4 r-4 -H 0: 0) -4 v-f 0D -4 v -f -H 0 44j I M

0- C) CD C D0 C) 7

--4 .. . 44 .4 . . -uC) . t fl.a) C)J -0 a)0 4

M44 +1 +H +1 ~ +1-4 +1 +14 +1 f +14 +1 +' +C+) C)

12 -- 4- 4 0S

-T 0- -H4 -1-'

4 4 4-4

44 VIO44 #J z. ' t4-4

C)u vi0' '-f II -C) I5-f -4 00 CDCD0 0ID 0

44i 00 . . .. . 41 0

C, 4- 44 4 I +1 +1 +1 I I +1 - -1 - C) ad -() 4)~ -- 4 0

Cd U I- LI-444 -4 -4 -4 H

E 4. . 4-4 .

144 0 44 -I tr

0 >- 1 0-4

*~~ 40 4- 400 0 -'4 4-4 0

4-J >' .- f(I) (AlCQ t- 00 r- *00 r t 00 f- .--T00 -f = -

.- 4 >,- -4 C1J (NJ r141. (NJ 44 t4 -4.0 c m 0-HO 0

54

Table 9. Analysis of variance significance levels for plasma aspar-

tate aminotransferase activity1

Plasma Aspartate Aminotransferase Activity(Unstimulated)

Vitamin Diet Interaction

Males

Day 7 .000 .001 .008Day 14 .059 .148 .375Day 28 .143 .765 .796

Females

Day 7 .002 .067 .11SDay 14 .062 .0442 .180Day 28 .349 .005 .036

Plasma Aspartate Aminotransferase ActivityCoefficient

Males

Day 7 .000 .000 .000Day 14 .000 .018 .536Day 28 .797 .794 .761

Females

Day 7 .001 .222 .029Day 14 .000 .000 .004Day 28 .000 .011 .045

1p values obtained by ANOVA on 5 food groups, 2 vitamin levels (Groups

C through L). 2Diet differences were not significant (P < .05) byNewman-Keuls test. Examination of 95% confidence .nlervals revcaledapparent differences.

551

0+1 +1 +1- -41 +1 +1- +1- +1 +1 +1 +1 +1I LO -1V) CD

TT4 "s ~r r- ~ -4r-- 10O mS- 0)ir- V 4J

4-) M. V)

r4 CDr l0 ) l 4 0'

0+1 + I I I + 1 +1 +1 +1 +1 +1I + 1 4 U

I- ('A 004HN D 0 -1t~ rl 4

0 * .H ~ ~ ~ ~ ~ 0 _;ts~ r-r S~-4 o-r-

o tnr4 r

C' 4 0O0 t 1 0 N.. Lo 00 C) m C) r- 3 70 i--r0- .. . C) r*l .O CD t' t- -4 r C'-1

+f. - [ + + 1~ x + +1 +1 X~ +41+1 +1 0 +,-+1l+l .0H0 0 -: jI

0 1- ) CD -j n'-4 C l C) 0 L0) T tCIO t- -. a) Ci

4J-C H. .. - . . .- 4-0.4 1:) C4t)r 0 $4 '* I 00 z ) 0

.14 0) 0- (u-0 0 0

a) C) in .0 0 0U) CIA no' vi LIS CN .0 4-0I u

C'S c'S r4 .' .'i~ .4 ,4~ n H iCD 1 ,--4 f1 '-4-40C, I l t I I C" 1'CI -

V/ ) 0 +1 +1 +1 +1 +1 +1 C/) +1 +1 +1 C/) +1 +1 +1 (4-0) -H4

z) 1/1 Cr5 Cf) C1)t-4 4 UCc L) a) -f r--,4 C) Le)00 ,4 -4 .D C) - -I L* i -4LO *H m -H4

0

$-4-H4 0 -4 . ' -4 .' . .C'S. . . -:$ r t-4 441 c 0 C'S LO vs C ) 14 0 (N' r0 N)'-i 04 LO E '"00 mQ

00 uLL. tn 'I 'ICI I r ) It CO ) 1:T ZIS:t . . -5 -r- +j- o 0H .H

7-1 Q0 C'CSS m -4>

C)V ) C) r0 - 00 t~ 'IT'~ o C 'S L -

C73 4-4 -4 CDn( '41) 1;' 0

z- j i 41 -41I-- +1 +1 - +I +1 + +1 -+ - +1 +1 4.):D.~ 1. 000C/ )0)C )C C'S -T

d/ -4 E LA 1; _ . .C4 .C4. _;I'

E C1)) 'TI - in- "t Tt in ~ SLOS *oW'C Vn L~-4.j

') .0 0 UHd! - 4- -4C

40 0-H +11-a C) (r, [-i") 00 10 0 Cd 4.4 c)

4-4 -4 1-4C)0 z 0 0 V)C O'St C ~

i -4 4! 1 1 1 +1 +1 -41 1 1 +1 -4- t-i C:

~ 0 ~- 1'I 2 C) -O

It-)) Lo 000~~~inr n~~-- C/C-

044 C) 00 C/0 .2 4-J 0)

414 -4 1 4-4C)) 1C) N-T 00 r- -I 0 1-~ TT C)r-' 0

-4 1-.- (1 1-C', .l -4CN .-I44.0 CI CL 4-4 r-, F--

56

cd Cd 4 +Iu ~-4 ('4L .I-- LnCd

.r40 000) C 000 00C)C0 C)000 i

Q) 41) 4-1-~u +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 41 r..-

(1) .1C)'- C' 0) C11 'IT C -t If'T0 nr t4) t)( Un

o Q) a0 C

u (n *H. +1

4J Q) 00

+1 +1+1 +1 +1 +1 +1 +1 +1 +1 +1 +1 -In ) 0

4j a) Wi - ) n t l t N -

z.H -: -: 4. -4 1+

-- 0 0 )00 0 0 04 000 - Q) .

0= -, a) C. .4 0 *r 0 CD0a) $4 *H *Ha). H+1 +1 +1 >4 +1 +1 +1 X +1+1 +1 0 +1 +1 +1 ~ 0

X0 0 "00 (1'I41) U 0 r-l* '0 -4 r-C: q00 m r-4-0 U) Lf0~ -I -4 CZ C)(r*H r ' T 1to to) .* n )H IlZt' .- ~t)h $-i k ~ ' 1fl 0n *" U 0

> 4-- pQ*H U ~. -I'

4- >-.-4. .'-4 - - a> r-4 +1 V)

CU 0 44Jt04L En V . .

a) 20 2 CD a)r4U 4) .0 0) If) V ) + 0cd -, iO cdC . C3 m C4) 11 U

*4 Ir CI C' 'J '\ II te)I N - r4 - 4 Nr-4r--1 +1 Va)H C'D 000 -4 00 000 000 C D CDC )44

4-4. .. . . .I . r- Z Itn 1ct Q) a)c

C0CL +1 +1 +1 +1 +1 +1 0n +1 +1 +1 V + +1 +1 a) :a4 -( 4Jco a) U), tn a) Q) r= I U r1.4-4 N a) oo -4 Ln) ( 14 o00 " -4 0CD -4 0'0) '.r---4 04J r= 0 '-4 te t0 -4 0) C ) C13 t to1~N co C'tn C4 CU1d NH0 ) $4 CU . * CU . .2 = I = a

0 ) LL .- 04~ ,-,4 ) v-4 -4 -4 a) r-4 -4 -4 4.j :3 -LL. -H. *' ) 1 0

4J 4l *H*, 0-=

* 'i > 4J' a) r-ra) u Cd .00 .- itU :- >

4J) Hej V)'4tn rI-4~ C'j".C' L/)'4A 4- CTS-4-) 1 CISas0 C44 000 C 00a0 000 00C )0 C> U

'j 0 cpi 0 a) %rC$41 ~ + +1 +1 +1I+i+1 +1 +I+1 + +1i+1 .C0.4.) -4 *P.a 0. a) C 4.' M.th r-4 I C'4'0uc ) p n000 -1 -4 -'T 4J - 0 r- -E

CU tn t) tn 'I VU) a)l an r )tl q) -4

CU -i ) - ~ 4-4-4 -- 4 4-4S Cd) 04-1 tf.4 .0(n to - A- CI4-40-

4.4 - a I (I r14 t) IN (N U) a)0 -4 000 C0 ) 0 + DCN 1-4

-H S54 a) V)14.44. I 1 +1 +1 +1 Ii I+1 +1+1 .14 VC 4-)

C $4 0) .0 ) s 0 C ) I- ., r- 11 -H4

u t) C'c (tn "' 44 '- -I. . . . . . 144 4 ) U

En a) 0 a44.4I- ) PNO (n

z~~ 0 D>u CU H W

Cm 0 4-) U 44i U-)0 -- 1-4 -.4 W

4J) > 4-4.t) (D r- t--T0 " 00 , c-t 0 r- h-'T 0 -4 H,-

'-4 -s4 .- 41NI -4 C' .- (N r-) -.0 00. U r- 4

CU0 C* 0V) .

57

Table 12. Analysis of variance significance levels for plasma alanineaminotransferase activity

Plasma Alanine Aminotransferase Activity

(Unstimulated)

Vitamin Diet Interaction

Males

Day 7 .000 .000 .679Day 14 .000 .007 .558Day 28 .000 .0262 .678

Females

Day 7 .000 .005 .252

Day 14 .000 .0122 .890Day 28 .000 .0463 .412

Plasma Aminotransferase Activity Coefficient

Males

Day 7 .000 .000 .024Day 14 .000 .004 .179

Day 28 .000 .407 .409

Females

Day 7 .000 .0302 .915Day 14 .000 .688 .323Day 28 .000 .501 .690

1P values obtained by ANOVA on 5 food groups, 2 vitamin levels (Groups

C through 1). 2Variances were significantly different by Bartlett's

test. Kruskal-Wallis nonparametric analysis revealed significant diet

effects at high vitamin level only. 3Diet differences were not signi-

ficant (P < .05) by Newman-Keuls test. Examination of 95% confidence

intervals did reveal apparent differences.

58

m 00 r4 C) Q - m0- t,0 N +1- -

4J1 + +I +1 +1 +1+1 +1 +1 +1 +1 +1 +1 4f) -

0- CD .1.4 LU o Cn \0 -4 N-O'0 mO' V

44 0. 4.'

03 a

000 4y.,- 000 -04'4M'--t t " 4 00 4-4

x - -4- +qN - -+4+4+41- +i+'+ r1 4-! C

0U -11

-4 bo ,A -..w () 00 C4. c''r'cl 0 00O Cl ) - 0) $-4

p. Q) 0 -4 C ) C) C) - . .4

a) r.4 xI.,.4 c -4 ,. +1 +(+ +1 + f +44- 1 +1 +1-f ) +d- '1 44 0

* -. 1 0 0 P..

0 Ln *- -4-4 -4 -0 - 0) 4r-- H4 V)+j4 .,- -40\ .. .0t' .. 4-CO Zt f U ) 0-> Q) 1 4 4 A ) NC C -4 r.f-4 Cj CL r~4 r L 41 t-r4 -14 0l. 06..4 -i +1-H

4 Ct 0) u i- $.cU 00 Lix 94 0

al, *o m m -4 C('4 l 0 t' 4) ('4 vt ,4N C ' -4 C>Lf) r- ~ 0

44.C Q) (d4

+1 + 1+1 +1 +j +4 +1 +1+1 tn + -4t! 44 -44-3C) .,I ChC 4- U U

$4) 'O )' =) -41- Ln LJ1n4 to -O ' -H Q q 0-4 .4

. . . CIS . . . .o4 4 v4-4. rC4 i r4 C IA (44' 0) ('4'C14 M0 "0 p

., 00'--o .t 0 0

C) C) Cd V)

*H-4 0 0 -4'm-40m (7 044 -t C ON 4 co C

= 14 4 (D+1+1 +1 +1 +1+1 + +1 +1 +1 +1 +1 . *-

-. 4-.

a ) 0) C4lC -1 -4 " r, (4 1 CI C)

tA~~U VC)j4

441 + I u U C(4.431 C). 4-4

0 (D V) 000 ~'-4 A 4 4q d)

. -H *,-- 4d 0 )

(45 4.144 44 4 + I + + t + I + 1 41 +1 0 4-4

1.4 0 ~-4 C1(\C' CCC) V)

0 4. U) 4

'-4 41 4-. 00j -4 r 4 Q)

U)C) ~ CO -~-C f-'50C4

59I

-H CT o V)L~O t),1 0nq 00 000 000 000 tn Ii r) ri

444-) $-4 4J..)u +1 +1 41 +1 +1 +1 +1 +1 +1 +1 +1 +1 u r-

-Q12 cn a.)-4+1-4 V) al '-4 0 1O nL U ru I 0t). 1-. -H4

Lo-*r4 ,HH C1 H r-4 -4 -4r-1 1 u -4

4-3 11

Cl .0. ) 4-4 C)C I )C D DC 0 -

14-4 u

Cn - -.t+ :t1+ +11+ -41+ M -4 Uqr-*.-)CJU)\ LAO) 4 ~ ~ 44- -H tJ-d

9 4-) E

.-H .H 1-4 to .cl..0

-1 'It -~ N ) I- )N 2 NCI LI)

Q) .- 0 0 2 0 2 0 . .ci +

0 CdH +f +1+1 X +1 +1 +1 X1 +1 +1 +1 0 +1 +1 +1 1-4 C-4Jf -0X. 1 0 0 Tl u 40 O(7 - . - o - ~,-N * )

4J~- tn *r bn - -4 r-4 tn . to t) N - -44 441k $H* 14 * * 14 . 4-) 0>

-- 4 -' r-4 > H -, H>~~ U1) *H t

0 0 0- cdc

1* 0n to CA .4-I Z UC ' n cq(

(1* 3U)-4' ~ ~ . N ~'-Z(4 0 0 0 G- 0 0 0 0 0 0 0 ts I)-

4-44+1 +1+1 +1 +1 +1 w) +1++1 v) +1 +1 +1rI-H $-4 U)U)) Q2) - 9

Nd 12 ) 'q- \0 ) (D) '- nU r-4 rq U) - _-4 v") 12)CO

0- 4 C ~ -A~ Ie-H 1 r4-4-) Id LL) r1 -4 -4 -4-4- -4 H -

O -Cd . . . 4-

.9-H -H 4+J 1)

Cd (U .-H u , c

'Hn U))n -4PU MN N-4 a >,a- 000 00C, 0 00 C 000 1 ~414-4

.- - . .2) .

C +++ +1 +1 +1 +1 +1 +1 +1 +1 +1 a;cd -4 -H

I~~~C 'tO) ~ -0 4-I 4.~ -~U)N -~~-4 -4C~. a -H

C~~12)~ -4) ,- 4rI-4414 ~ +1

-4 -HU) cs )tO 0.

-4 $4 V N 1-I

0 -H -H -4 000 000 CD~ CD-C)Cl

W- o0 C) 1- Ln C

-H) 1-4 02 -'2

00~4- 00. P4) - H-44 o :sN ' 4 -4 41 - -

0 -q L2nH

60

Table 1S. Analysis of variance significance levels for crythrocytcalanine aminotransferase activity

1

Erythrocyte Alanine Aminotransferase Activity

(Uns timul at ed)

Vitamin Diet Interaction

Males

Day 7 .000 .000 .000

Day 14 .000 .000 .000Day 28 .000 .000 .000

Females

Day 7 .000 .000 .067

Day 14 .000 .000 .003Day 28 .000 .000 .025

Erythrocyte Alanine Aminotransferase Activity(Coefficient)

Males

Day 7 .000 .004 .000Day 14 .000 .021 .012Day 28 .000 .004 .054

Females

Day 7 .000 .283 .362

Day 14 .000 .397 .540Day 28 .600 .429 .867

IP values obtained by ANOVA on S food groups, 2 vitamin levels (Groups

C through L).

61

cd Cfs.0 .0 mU .0Q. 0 -

000/)r L) 00- 000 0o00 0 I0 C00 000 000 000 CV 43D

4.r,00C C )C> C 0 C) CDC) V -

CU ta. r-)4Ji 1 I 4 H + 1 +1 +1 + I + 1 +1 41 + 1 --- 4

4.)0 't~- ~ C"1' 0l0 Cot- IJnr- 14 it)3I)0'-4' ID - -4~- r, O)t -4444) C)--4.-4 .- 4 " " <D-4 -1 -4 1-1 4 4.3 V

-4 . .LI . . .0 . . . . . .0 . .

00 -40 00 000 C) C cC U000 00)C C )0 00Co0 00 C)C C

x41+1+1 +1 +1 +1 +1 +1 +1 +1 +1+1 Wo 1 n 0 14 c -0t-I C) Co4r"

-4 '0 0 -1- 14 ('J1t' CU 411 -4 40 C* . * . V)1 0:

C .0 Cd Cd m 1 l0 C l - --- 4 th CI d "~ ( 10C 00 "T C)00 -.. C.IJN CI - ) 'o r-0 s-r- +I

CD) ~ . 00 0 a) C) -4 0 C)000 C) 0v-4 tU a)U 0 00 r- C000 CD 0 000 ( _ -

.14 .* . -4 u 0 I)0> 3 +1 +1 1 0 +1 +1 +1 0 +f +1 +1 +1 -- l t+1 ) 044

04 -'- :3 c

OV (*r4 Nr'- tl 71 4 " 0'0 $.4 k It0 'j C)Ln 1cli CU iD 0.-1-4 -4 r4C') 0.-4N 0. -4f' C -4 +j =4-'

4-~ 0CU ++1+1 1+1+ 3/I 1+1+ 111 1+c+ *-.o '

CU 0.-'s- CU Cd'J- (440M 4,4'4-4 CL. *. .n z . \0 *. " 0t -t C .fEA. o .) 4 ClC C14 0 3C ) 0c.jo

r_' 0 A C)0 CD - 0 C C ) U~0 . C C

p F4 4-.4 CN ' C -o0I CC 400 000( 0 00 00WC

tU 4) \.4 000o (1 000 - - 00 000 "4.3.t-

o) -4 3/ICn)4 V o i n 4 C3 -41- V$- . +1 4 -4 t-I fH r= a1+ -4 4-I +1 4 -I4I f, 41 '-4

C% .4 cz 4-- It-. 0 oN) If N C4 04 0

o~~~~~ CU =.'O OL1 N tI ~ -

U- -4 00.- CD C) 0 0 0 0- C) ~ 4 CU 0 CD 4 )

.~ ..4 .. . .

'-4 co 1- 0O nr' )t, :w.0 C)/ 000 000 -4 14'4 0

V). . . . . . cz 0 .C4JL 0 a) 4-

a)C OQi 4 CU 0 .Q44- :m00 -i- 4 10 00 0.U -

0. () )0) 140 Co0 0) 0

0 . . . * * * .C

$.4- u # 0

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L" 0- 4-. 0p .0 .4.

41 r

.04 CA0 -4 Cf3 -4 HqLCd 0 -4000 t

62

It)

Cd Ed C +104 4 -I -4 ,--44 r~-4 -4-4 1

+1- .)+1 +1 +1 +1 +1 +1 +1 + * +

'-44-4

000 000 .0 000 C -

S- -4 -4 14 4 -4 -4 '-4 .4-1 -4 .- 4 r4 C:

-4~ .-(3C' CD a 0000CDC )!

*') 00 0 000d*

=) '-(1 \O0 Ln r- An 'T t~- L) \0 Lt) M

04rI0 00C)0 00 000 C) oCD

0 CD 000 C> 000 V 00 C 00r-C0 N

.r. =*q +1 +11 + 1 +1 +- ) +1 +1 +1 0 +1 + +1-I N+i>0 v- x 0 4)r At0 CIA Z 00 ~tn M '0 (N r- 14) *-4 '3 10N P 4)0 r

4 o "~ a4 C) 000 H,- ~40 C) 0pCDo0 Z Hu 0 *,H . * * 4 . . . N . . o '0

Nd r-4 .- 4 -4 >, -4 '4 ~-- 4 -l ,-4 CL. -4 -4 -1-4 4

N- .H 0

Li') $- r= C) Ln44 0 0

U) r -4 V- - ' - I- 4 -4 4 rI -I '4 *H= I 4 C~0O Z:) 4 00 0 0 0 0C)0 -0 1

4V+1 +1 +1 +1 +1 +1 U) +1 +1 +1 tA +I+1Ii 4- ) V- -.

4) Q -: V Iq -' 0 -4 'O D d 'q0 .- -40 ,-4 D000 csCI5 000 cis 000 C -4

Nd .,I LL. * .. 0)1

(D 0 -

N r-4 -4

Nd ..

QVV +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 W44

0 r-0~ -'0 V4-

NH V1 -4 4. -4 -44.-4,-4 -4 14 1.4 ' '-4 0'Cd, '-~ 4-)

4 J~ :31 :

0U 4) ci

44 CO rV4 1-4 1-4 V) 44ON *4-,A-4 000 0M,0 -4 Q)

0 0 . . . . . .

1..4) I +1 +1 +1 I +1 +1 +1

.0

En-4 0 041.,

4 .04 1. ..

14> 4 C4 ,-4 r-4 C1 4 CIA t4-4 .-.0 C Q. 44

d .4 -4 0)

-. .

OFFICIAL DISTRIBUTION LISTComander Director

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('mmander CommanderU.S. Army Natick R&D Command U.S. Army Natick R&D CommandATTN: U.S. Army Rep/JTS ATTN: U.S. Air Force Rep/JTSNatick, MA 01760 Natick, MA 01760

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66