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~ SAX-TR-66-21

POSTIRRADIATION CREATINURIA IN

iMACACA MULATTA PRIMATES

I. G. PETERS, M.A.

H. E. HAMILTON, B.S.

top. FEDERAL SCIENTIFIC ANDS-TECHN.C.AL, INFORYLAT1ON -_

~ I~ August 1966

USAF School of Aerospace MedichieAerospace Medical D1ivision (AFS)-

Brooks Aar Force Base, Texas C

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POSTIRRADIATIOK (REATINURIA IN MACACA MULATTA PRIMATES

LG. PETERS, M.A.

I H. E. HAMILTON, B.S.

FOREWORI)

This report was prepared in the Radiobiology Branch under tan,• No. 775702. Therevised paper was submitted for publication on 2 June 1966. The work was acconi-plished between June 1965 and January 1966.

The experiments reported herein wete conducted accoiding to the "Principles ofLaboratory Animal Care" established by the National Society foz Medical Research.

This report has been reviewed and is approved.

AMES B. NUTTALLColonel, USAF, MCCommander

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ABSTRACT

Mlacaca mulatta priimtts irradiated b, Co'," gamma rays in three groups of fouraninmals each to dose 'evels of 2,000, 4,000. and 6,000 rads showed marked creatinuria.

Refinement of the fluorometric det.ermination of creatine based on the reaction ofninhydrine with creatine in a!kaline media has been achieved. Interfering guanidocompounds calculated on a creatine ?quivalence constituted from unmeasurable amountsto approximately 1014, of the total creatirte in samples containing a small amount ofcr'eatine.

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POSTIRRADIATION (REATINURIA IN MACACA MULATTA PRIMATES

1. INTRODUCTION were confined in metabolic cages throughoutthe course of the experiment.

An earlier study from this laboratory estab-lished that Meicaca mldatta primates exhibit a Samples were refrigerated or frozen im-postirradiation creatinuria (1). Four Macaca mediately after collection when determinationsmulatta primates on a creatine-free diet showed were not made immediately. The frozen sam-a marked increase in urine creatine after 1,000 pies were thawed just prior to use, and ther!p of filtered 250 kvp x-ray. remaining solution was refrozen for a duplicate

determination.The purpose of the present study was to

extend the initial observations to higher irradi- Creatine was determined by the fluoro-ation dose levels with Co"" gamma rays. metric method of Conn (2). A model 110

Turner fluorometer equipped with the general

H. MATERIALS AND METHODS purpose primary and secondary filter pair(<4000 A. primary; > 4100 A. secondary) was

I Twelve normal adult Macaca mulatta pri- used for all of the analyses. Quinine sulfateTwele nrma adlt acaa iulata ri- and creatinie standards were run daily. All;mates, composed equally of males and females, asurement s were r ragent bla

were randomly divided into three equal-number fluorescence.groups. They were fed a normal diet. Onegroup was irradiatet: with 4,000 rads (415 In four determinations on urine from post-

* rads/min.); the second group was irradiated irradiated animals, the order of mixing thewith 6,000 rads (415 rads/mir.) ; and the thirdgareagents was reversed. The diluted urine wasSgroup was irradiated with 2,000 rads (200 added more than fivc minutes after the admix-rads/min.) after serving as a baseline control adde or tha fiv mntes h e a ix-

j! for two days. To supplement the two-day base- the nndin olion.

line control group, a baseline was established the ninhydrin solution.

for an additional group of two males and two In four other determinations, 0.1 ml. of con-females for four and one-half days. Also, four centrated hydrochloric acid was added to 1 ml.

i:animals were sham-irradiated.am we a ideof the urine, and the mixture was digested in

The irradiation was given in two equal doses a water bath at about 100° C. for three hours.No pH adjustment was made before proceedingi• of ventrodorsal and dorsoventral exposure to wi h heC n m t od n e ai -r a ed s -

giea more uniform dose-depth distribution, with the Conn method on the acid-treated sam-.• give a m r unf r doed ph dsrb t n, pies; however, an equal volume of acid was

A one-minute interval interruption of the ir- Sincorporated into the reagent blank.radiation was required for repositioning theprimates. The irradiation dosages are on an

'•' animal midlin:! dose basis. I11. RESULTS AND DISCUSSION

Urine samples were collected twice a day The results agree with previous observa-• except for a single two-day period when they tions on Macaca mudatta primates; the animals

were collected once a day (table I). The animals at all three dose levels exhibited a general

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TABLE I

Creatinuria in Macaca mulatta primates irradiated with cobalt-60gamma rays

2,000 rads

Day Animal T43 Animal V92 Animal V42 Animal V66Conc.* Ratet Conc. Rate Cone. Rate Cone. Rate Av. rate

1.0.04 r. 0.06 0.32 0.30 1.15 0.18 1.02 0.650.0( 0.00 0.04 0.22 0.88 3.40 1.21

1.68 8.19 0.42 1.78 '.46 9.87 1.48 6.06 6.482 2.60 11.03 0.65 6.25 1.80 13.71 1.40 3.85 8.71

3 2.00 6.05 0.40 1.95 1.04 3.20 1.86 5.34 4.144

0.44 5.43 1.30 5.36 1.56 4.56 5.125 0.36 5.54 0.94 6.51 1.00 4.62 1.60 6.40 5.77

1.30 4.38 0.98 3.30 2.60 4.19 1.16 1.66 3.386 0.01 0.02 1.00 3.49 0.28 0.7b 1.80 3.64 1.97

7 2.20 1.04 0.66 4.943.90 5.72

8t__ _ __ 2 __ _=,_ _

4,009 rads

Animal T45 Animal T92 Animal T83 Animal S71Conc. Rate Conc. Rate Cone. Rate Cone. Rate Av. rate

1 .68 3.85 0.42 2.47 0.70 3.66 0.52 5.34 3.831.25 6.36 0.65 4.77 0.34 0.91 0.44 2.08 3.531.46 7.10 -0.55 3.16 0.19 0.42 3.56

2 1.41 5.67 1.95 5.73 0.68 4.80 0.09 0.22 4.10

1.12 3.10 1.30 5.39 0.70 6.23 0.04 0.09 3.703 1.20 4.76 0.35 3.79 0.48 5.62 0.05 0.15 3.58

1.38 5.25 0.90 5.11 0.70 5.45 0.Oi 0.03 3.961.60 11.89 1.40 8.53 1.20 5.66 8.69

5 0.80 -2.71 0.80 2.71 0.74 3.68 0.16 0.45 2.39

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7 1.22 2.156,000 rads

Animal V90 Animal T90 Animal S47 Animal S41Cone. Rate Cone. Rate Cone. Rate Cone. Rate Av. rate

0.22 1.44 0.12 0.57 0.68 1.74 0 27 1.26 1.250.00 0.00 0.00 0.00 0.00 0.00

0.70 2.79 0.00 0.000.92 2.88 0.00 0.00

1.38 2.821.80 5.611.90 3.06

0.00 0.00

5 1.04 2.13

6OConcentration of creatine is expressed in milligrams per milliliter of urine.MRate of creatine excretion in the urine is expressed in milligrams of creatine per hour.JA urine sample was taken, but nn analysis was run on the sample.

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crea. 1Tu Tk onnt, r,, ., ,id (.3) The alkali-catalvzed opening of theanimals excreted at a rate (mean rate of five-nmembered ring of ninhydrin is rapid. The0.40 mg. creatine/hr.) well below that of the o-carboxyphenylglyoxal conversion to o-car-irradiated animals (table II). Sham-irradia- boxymandelic acid is relatively slow, requliringtion, in general, had no effect (table III). about five minutes.

One marked difference between the re:;uits The molecular species reac.ing with creatineof Krise et al. (1) and the data reported here is presumed to be o-carboxyphenylglyoxal.is that approximately a tenfold difference in Because of the transient nature of o-carboxy-the postirradiation creatine level is evident be- phenylglyoxal, fluorescence is not developedtween the twc' experiments. This difference unless the guanido compounds are present frommay be interpreted on the basis that a creatine- the initiation of the reaction sequence. Wefree diet was used in the previous study, have taken advantage of this fact in preparingwhereas a normal diet was utilized in the a urine blank. Diluted urine samples (1:20,000)present study. added five minutes after the admixture of the

alcoholic potassium hydroxide with the nin-The creatine procedure of Conn entails the hydrin solution gave no evidence of fluo-

conversion of ninhydrin (1,2,3-indantrione rescence over that of the reagent bhnks. Thishydrate) to o--carboxyphenylglyoxal (2, 3). observation, in addition t.- the fact that theThis a,[3-dicarbonyl compound is unstable in preirradlation control samples all exhibitedalkaline media and undergoes an internal Can- negligible fluorescence, indicates that i he onlynizzaro reaction to give o-carboxymandelic fluorescent materials being detected by the pro-

cedure are active guanido compounds.

F "-x2000R To estimate possible interference with theIl --- 4pooR creatine determination by other active guanido

8- SHM-.6000RR compounds, several urine samples were pre-SSHAM IRRADIATED

-CONTROL MEAN treated with hydrochloric acid on a water bathI- for thre2 hours. According to Folin (4),creatirne is converted quantitati--.,ely to creati-

6 nine by heat and acid. Creatinine is a N,N'-substituted guanido compound and does not

• 5 -'\ yield a fluorescent species with ninhydrin inEz strong alkaline solution. Other suspected inter-S,,fering guanido compounds such as arginine are

relatively stable to the acid treatment, thusW permitting an estimate of the amount of their

3 -• interference. Interfering guanido compoundscalculated on a creatine equivalence constituted

2 - X from unmeasurable amounts to approximately+

1 0c'/ of the total creatine in samples containinga small amount of creatirie. No correction was

A /made for this interference.2 3 4 5 6 7

DAYS Duplicate determinations separated by atime interval of several weeks revealed nomarlked change in creatine content of the sam-

FIGURE 1 phes with time. Alsr, control creatine samples

The rate of creatine excretion in the urine after show that creatine is stable in frozen aqueousirradiation, mixture for a period of several weeks.

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TABLE II

Creatine cont? 91 values

Animal T43 Animal V92 Animal V42 Animal V66Day Cone.* Ratet Cone. Rate C3nc. Rate Cone. Rate Av. Rate

0.20 1.33 0.08 1.24 0.13 1.27 0.02 0.28 1.03

0.08 0.27 0.05 0.77 0.05 1.83 0.02 0.88 0.94

0,02 0.09 0.02 0.29 0.06 0.71 0.05 0.54 0.41

2.00 0.00 0.02 0.16 0.00 0.00 0.01 0.70 0.22

Animal OB8 Animal 7B4 Animal W59 A nimal V41Cone. Rate Cone. Rate Cone. Rate Cone. Rate

0.12 0.75 0.05 0.25 0.08 1.27 0.03 0.22 0.62

1 0.00 0.00 0 07 1.60 0.01 0.25 0.00 0.00 0.46

0.06 0.20 0.05 0.34 0.10 0.63 0.04 0.24 0.352 0.09 0.02 0.03 0.15 G.11 0.08 0.06 0.38 0.16

0 11 0.04 0.09 0.37 (619 0.68 0.09 0.25 0.333

0.06 0.17 0.91 0.00 0.03 0.49 0.07 0.20 0.21

0.01 0.02 O.O0 0.00 0.04 0.39 0.02 0.10 0.134II0.00 0.oUo 0.05 0.44 0.03 0.13 0.05 0.13 0.17

0.02 0.21 0.03 0.19 0.02 0.18 0.03 0.09 0.17

Concentration of crezitne is expresseid in milligrams per milliliter of urine.

?Rate of crentine excreticn in the urine is expressed in milligrams of creatine per hour.

TABLE III

Effect of sham-hiradiation on creritine excretion

Animal 0B3 A nimal 0B7 Animal W83 Animal 8D2Day Conc.n -z. R Conn. Rate oCoic. Rate Av. rate

0.26 1.58 0.22 0.92 0.11 0.64 0.01 0.07 0.800.01 0.3*2 0.00 0.12 0.01 0.69 0.00 0.26 0.35

0.01 0.04 0.16 0.21 0.17 0.51 No rtrine output 0.19

0.01 0.12 0.08 0.25 0.11 0.71 0.01 0.30 0.34

0.01 0.23 0.05 0.47 0.12 1.26 0.01 0.11 0.52

0.01 0.29 0.01 0.41 0.03 0.93 0.00 0.08 0.43

0.06 0.46 0.06 0.23 0.46 1.62 ' 0.01 0.02 0.580.01 0.39 C 02 0.17 0.10 0.66 0.01 0.10 0.33

0.02 0.19 0.08 0.09 0.02 0.14 0.01. 0.07 0.12

0.01 0.8c 0.00 0.65 0.31 0.75 0.00 0.31 0.64

6 0.04 0.26 0.07 0.14 0.04 0.07 0.02 0.06 0.13

0.01 0.36 0.03 0.20 0.00 0.12 0.01 0.05 0.18

7 1 0.69 2.38 0.38 3.42 0.23 0.98 0.02 11 1.72

Llouh!e line indicatte. hatrr-iri dliation.

*Concentrationl of creatine i, vxvit.•kl in milllgianes per milliliter of ur.ne.

;Ra.e of creatine excretion iii the ui iJoe ib e\lkrvsm..l ilk milligram.. of reeatine pei hour.

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REFERENCES

1. Krise, G. M., C. M. Williams, and D. R. Anderson. 3. Ruhemann, S., Jr. Triketohydrindine hydrate.Postirradiation creatinuria in Macaca mulatto. Chem. Soc. 97:2025-2031 (1910).Pioc. Soc. Exp. Biol. Med. 95:764-766 (1955).

4. Folin, O.,Jr. On the determination of creatinine2. Conn, R. B., Jr. Fluorimetric determination of and creatine in blood, milk, and tissues. Biol.

creatine Clin. Chem. 6:537-548 (1960). Chem. 17:475-481 (,914).

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Macaca mulatta primates irradiated by Co6 0 gamma rays in three groups offour animals each to dose levels of 2,000, 4,000, and 6,000 rads showed markedcreatinuria.

Refinement of the fluorometric determination of creatine based on thereaction of ninhydrine with creatine in alkaline media has been achieved.Interfering guanido compounds calculated on a creatine equivalence constitutedfrom unmeasurable amounts to approximately 10% of the total creatine in samplescontaining a small amount of creatine.

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RadiobiologyRadiation effect in primatesPrimates, Macaca mulattaCreatineCreat inuria

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