The effects of selenium on the metabolism of methionine in sheep.

The effects of selenium on the metabolism of methionine in sheep Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Oregon State University on 01/01/15 For personal use only.

M. HIDIROCLOU~ AND C. G. ZAKKADAS' Animal Resectrcll I~lstitlrterrrzd Food Re~earchInstitzrte, Carlada Department of Agricl4itlrre, Ottonw, Ont., Car7udts K I A OC6 Received April 7, 1975 H I D I R ~ G LM., ~ U and , ZARKADAS, C. G. 1976. The effects of selenium on the n~etabolism of methionine in sheep. Can. J. Physiol. Pharmacol. 54, 336-346. Two groups of sheep fed a diet of hay known to produce nutritional muscular dystrophy, one group of which received selenium supplementation, were used to study the effects of selenium on the metabolism of administered I.-[""Sjmethionine by runlen microflora. Rumen bacterial proteins of the Sc supplemented sheep contained significantly higher levels of radiosulfur than the bacterial protein of the non-supplemented sheep. Of the L-[.'%]methisnine present in the rumen liquor samples from Se-supplemented sheep 2 h after administration, 13.3% of the amino acid, which was measured as methionine sulfone, was found in the microbial proteins. A large proportion of the administered labeled methionine was resynthesized as cyst(e)ine which may account in part for that determined as cysteic acid in rumen bacterial and plasnla proteins. The observed low levels of radiosulfur found in rurnen microflora from selenium deficient wethcrs, indicates that the presence of selenium profoundly affects the rate of methionine metabolism and the distribution of methionine in rumen bacterial and protozoal proteins. In another experiment, the effect of selenium on the metabolism of I.-[Me-"Hlmethionine was studied. The selenium status of the sheep had no significant effect ( F 0.05) on the distribution of " S radioactivity in the blood plasma and tissues.

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H I D I R ~ G M. L ~ et ~ ,ZARKAD~S. C . G. 1976. The effects of selenium on the metabolism of methionine in sheep. Can. J. Physiol. Pharmacol. 54,336-346. Le but de cette ktudc a kt6 de connaitre le mttabolisme dans le rumen d'une dose de L-["%]mCthionine administrte directement dans ce compartiment gastriqile dl1 mouton trait6 ou non au stlCnii~m.D e i ~ xlots d'animaux, l'un trait@au s ~ l t n i u met l'autre non traitd, ont dte nourris pendant environ 10 mois avec un foin provenant d'exploitations atteintes de myopathie. Des radioactivitCs m~ptrieuresont t t i enregistries dans la microflora du rumen du mouton trait6 ail sdlinium, comparativement ail non traitt. Le taux d'incorporation de la radiomdthionine dans les prottines bactkriennes dl1 rumen de rnouton trait6 ail stltnium a ttC de l'ordre de 13.396, et s'est retrouvte sous forme de mtthionine sulfone. Une certaine proportion de la radiomtthionine cataboliste a it6 resynthitisee comme cyst(e)ine et mkthionine, lesquelles d'ailleurs ont it6 identifiies en partie comlne acide cystiiqi~edans les protkines bactCriennes et plasmatiques. 1-e taux inftrieur d'incorporation de radiomkthionine observe dans la rnicroflora du rurnen chez lc mouton non traite, dtmontre que la prtsence du stltnium a line influence profonde sur le taux d'incorporation de cet amini soufr6 dans les protiines bactkriennes et protozoaires. Dans une autre exptrience, on a itudit le metabolisme de L-[Ale-W]methionine chez le rnouton trait6 011 non ail sCltnium. Si une tendance gintrale B plus de rktention de radioactiviti. tissulaire et it molns dans le plasma s'est manifestte chez le mouton trait6 au stl6niun1 comparativement 0.05). au non trait6, cette difftrence n'a qiiand m6me pas t t t significative ( P

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Introduction Selenium is an important dietary factor for the prevention of nutritional muscular AsBREvIanoNs: BW, body weight; TCA, trichloroacetic acid; dpm, disintegrations per minute; Se, selenium; TDN, total digestible nutrients. 'Contribution NO. 573 from the Animal Research Institute. 'Contribution No. 243 from the Food Kesearch Institute, Research Branch, Canada Department of L4griculture,Ottawa, Ontario, Canada K I A OC6.

dystrophy, particularly in cattle and sheep (Jenkins and Hidiroglou 1972)- However? much concerning the function and metabolism of selenium and the biological interactions between sulfur and selenium in ruminants and rumen nlicroflora is not known. In ruminants dietary selenium is incorporated into rumen microbial proteins as selenometllionine selenocyst (e) ine, and subsequently appears in the and fluids the host (Hidiroglou et al. 1968, 1971, 1974). In cer-

pssues

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Oregon State University on 01/01/15 For personal use only.

HlDlROGLOU A N D ZAKKAI9A.S

tain strains of bacteria and yeasts it has been demonstrated that selenium may be metabolized via similar enzymatic routes as sulfur (Shrift 1961). Cohen and Cowie (1957) and Tuve and Williams ( 196 1 ) demonstrated that Escherichiu coli is capable of synthesizing selenium analogues and their metabolic intermediates of sulfur amino acids and incorporating them into bacterial proteins. The sulfur atom of S-adenosylmethionine also can be replaced by selenium (Mudd and Cantoni 19579 and some properties of the selenonium derivatives in yeast have been described (Mudd 1965, 1970; Cantoni 1953). From kinetic evidence, Tuve and Williams (1961 ) indicatcd that selenium and suIfur are competitive for the synthesis of cyst(e)ine and methionine and the corresponding seleno analogues in E. coli. Huber et ul. ( 1967) suggested that E. coli K-12 is capable of metaboIizing inorganic selenium only when 60-70% of the sulfur normally required for microbial growth is present. Venkov and Stanchev (1969) found that selenium supplementation to lainbs enhanced the absorption of L-["Slmethionine from the digestive tract. Lazarov (19681, on the other hand, could not demonstrate changes in L-[x?3]methionine absorption as a result of selenium supplementation in the diet of chickens. The present in viva investigation was undertaken to study the effects of selenium deficiency and supplementation on the metabolism of rumen microflora following oral administration of labeled methionine to sheep. The extent of distribution of labeled methionine in microbia1 proteins, and in the tissues and fluids of sheep was also determined.

337

sulfone, L-cysteic acid monshydrate, DI--homocysteic acid and I.-homocystine were obtained from Schwarzi Mann, Orangeburg. New York. and Beckman type I standard amino acid calibration mixture from Beckman Instruments Inc., Palo Alto, CA. All other chemicals were reagent grade and were used without further purification. A rzirlltrls trrld Diets

Sixteen 1-year-old Leicester wethers were used in these experiments. Eight of these animals at 2 months of age were randomly selected as a control (group 1 ) and fed trd lihitzlrn a diet of selenium deficient hay from northern Ontario where nutritional muscular dystrophy is common. The selenium level in this diet was found by analysis, using the method of Hoffman et ul. ( 1968), to be 0.02 ppm Se on a dry matter basis. Because the chopped hay fed was low in both digestible energy (45 % TDN ) and crude protein (7 5 1 ), sucrose and urea were added as supplements at level? of 6%) and 296, respectively, by spraying a water solution of these nutrients over each day's allotment of hay during its preparation. The remaining eight experinlental animals (group 11) after weaning ( 2 months old) were fed an identical diet, except that they received intran ~ u s c ~ ~ l a rdoses ly of 6 mg of selenium as sodium selenite solutions into the gluteal muscle at monthly intervals. The sheep were allowed to drink tap water trd lihitzlm. All animal5 were healthy and free from any clinical symptoms of nutritional muscular disea5e. After 10 months of this diet two experiments were carried out. Experir?lenl 1 In this series six animals were used, three from each of groups I and 11. In each trial, one pair of sheep of equal weight (45-55 kg), representing each treatment group, were dosed intraruminally by stomach tube with 5.5 pCi of L-["Slmethionine per kilogram HW. Commencing 1 h after dosing, blood samples (10 ml) were collected from the jugular vein at 1-h intervals for 8 h and then once more after 24 h with the usual precautions described by Perry and Hansen (1969). The blood samples were transferred quantitatively into centrifuge tubes containing heparin (0.05 ~ n l , 1 % ( w i v ) in saline), were centrifuged at 10 000 x g. the cells were removed and then an equal volume ( 10 m l ) of 20% TCA solution was added to serum. The tubes were then centrifuged at 2000 x g for 20 rnin at 2 "C, Materials and Methods the supernatant and sediment were separated and the amounts of L-["qinethionine incorporated in the Muterials L-[Me-W]meththionine (7 pCiimmol) was purchased protein-bound plasma components and in the TCAin aqueous solution from the Radiochemical Centre, soluble fractions were determined. Samples of rumen Amersham, England. I.-[""SJhiethionine (320 p C i i contents (100 m l ) were collected by means of stomach mmol) and PCS (phase combining system) solubilizer, tube 2, 4, 6. 8 and 24 h after administration of the a liquid scintillation mixture, were obtained from radioisotope and were strained through four layers of AmershamiSearle Corporation, Arlington Heights, IL. cheese cloth. 'The protozoal fraction was prepared from Omnifluor. a crystalline scintillation mixture containing portions of the clarified rumen liquor by repeated washings with bicarbonate buffer solutions and centrif98% PPO (2,s-diphyloxazole) and 2% bis-h.ZSB, and Protosol, a 0.5 M quaternary ammonium hydroxide ugation as described by Abou Akkada and Howard d solution, used as tissue solubilizer, were p ~ ~ r c h a s efrom (1960). The bacterial and cell free fractions were New England Nuclear, Dorval, P.Q. The sulfur amino prepared from other portions of the rumen filtrate, acid standards, L-rnethionine, L-methionine-DL-sulf- previously freed from protozoa and plant material by oxide, L-methionine-DL-sulfoximine, DL-methionine centrifugation at 600 g for 2 min. These fractions were


338

CAN. J. PHYSIOL. PHARMACOL. VOL. 54, 1976

then purified by repeated centrifugation (20 000 x ,q, 4 "C, 30 min) and washed according to the procedure described by Wright and Hungate (1967). Washed bacterial suspensions consisting of one part wet weight and four parts water were subjected to ultrasotlication in a 20 Kcis sonicator (model W-185G Sonifier Cell Disruptor. Harvey Instruments, N. Tonawanda, N.Y.). The ultrasonically-disrupted bacterial cells were then treated with 20% TCA and the precipitated bacterial proteins were freed of TCA by repeated ether extractions (Hidiroglou et a / . 1974). All rumen liquor fractions, washed protozoa, bacterial proteins and cell-free extracts, were frozen and stored at -20 "C for subsequent analyses

Experirttrtat 2 Ten animals were used in this series, five from each of treatment groups I and IT. In each of the five trials, two sheep of equal weight (one from each group) were given an oral dose of 16 pCiikg RW of I,-[Age-"HImethionine. Five days after dosing, the animals were killed and various organs and tissues were sampled. All samples were freeze dried and stored at -20 "C for radioactivity determinations. Anulytical ~Metlzods Anlirao Acid Arzalyses Amino acid analyses were done either on a Beckman Spinco model 120B or on a Jeol model J 1 C-5AH amino acid analyzer equipped with an LKB UItrorac fraction collector (LKB-Produkter AB, Bromma, Sweden). Triplicate samples of blood plasma and the various fractions from rumen liquor were analyzed for nitrogen by the micro Kjeldahl method (Steyermak 1961 ). Identical samples, equivalent to 1.0-3.0 mg of nitrogen, w-ere hydrolyzed under vacuum in constant boiling HC1 at 110 "C for 24, 48 and 72 h. respectively, with the usual precautions described by Moore and Stein (1963). The HC1 was removed under vacuum in a Rotary Evapo-Mix at 40 "C, the residue was taken up in p H 2.2, 0.2 hf citrate buffer, and the basic, acidic, and neutral amino acids were determined from a diluted sampIe containing 0.1-0.3 mg of N by standard procedures of amino acid anaIysis. However, methionine and cyst(e)ine were determined separately as their oxidation products by the performic acid procedure of Moore (1963). Triplicate samples of the various rumen liquor fractions containing 1.0-5.0 mg of N were transferred into Pyrex glass tubes ( 18 x 150 mrn) and were dried under vacuum. Depending on the amount of protein to be oxidized, 2-5 ml of performic acid solution generated by an admixture of 19.0 ml of 98% ( v j v ) formic acid and 1.0 ml 30% (v/v) hydrogen peroxide was added to each tube. The oxidation was allowed to stand at 0 "C for 18 h, then 0.30 ml of 48% HRr was added to stop the reaction and the mixture was dried under vacuum at 40 "C. The samples were then hydrolyzed in ~.lzcuoat 110 "C for 24 h with 3-5 ml of constant boiling HCI, the acid was removed under vacuum as before, and the final residue was taken up in p H 2.2, 0.2 M citrate buffer. Samples containing 0.1-0.3 mg of N were chromatogrammed on

a standard amino acid analyzer using a 0.9 x 55 cm column packed with Beckman type AA-15 cation exchange resin operated with citrate buffers ( p H 3.28, 4.25) at 6 ml/h. Cysteic acid and methionine sulfone emerged off the column at 21 and 52 min and were quantitatively determined by absorbancy measurcments at 750 nm and 440 nm in the amino acid analyzer with the ninhydrin reagent which was pumped into the effluent stream at 30 ml/h. The eluant was collected in 2.0-ml fractions. Fractions were then monitored by radioactive counting of 2.0-ml samples in a Reckman LS-250 liquid scintillation spectrometer. The rccovcry of cystine plus cysteine as cysteic acid and of methionine as the sulfone was calculated relative to the yields obtained by the performic acid treatment of standard solution of these amino acids.

Measurernrnts o f Rodionctivity Triplicate determinations on aliquots of the various fractions from the rumen contents, plasma, and TCA supernatants were assayed directly for radioactivity using 10 ml of PCS-solubilizer scintillation fluid. The effluent fractions (2.0 ml) from the ion-exchange column of the amino acid analyzer were transferred to scintillation vials, 10 ml of PCS scintillation solution w-as added, and their radioactivity was determined. The radioactivity in each sample was monitored in a Packard model 3320 liquid-scintillation spectrometer using the autorrlatic external standard to correct for differences in sample counting efficiency. In the case of solid fractions from the rumen contents, triplicate samples equivaIent to 30 mg of N were dissolved by heating the sample overnight at 40 "C in 2 ml Protosol solubilizer, and 10 ml Omnifluor scintillation fluid was then added. The samplcs were assayed for radioactivity as described above except internal standards were used to correct for differences in counting efficiency. All isotope data for radiosulfur activities were corrected for decay to the corresponding midinfusion dates. Triplicate tritiated tissue sampIes (50-60 mg) were transferred into scintillation vials and were then dissolved with 2 ml Protosol soIubilizer by heating at 50 "C overnight. Fifteen millilitres of Omnifluor scintillation fluid was added and the samples were counted in a Beckman LS-250 liquid scintillation spectrometer using internal standards to correct differences in sample counting efficiency. The counting efficiencies of and "H samples were 20-80 and 15-35%, respectively. Statisticab A nnl-ysis In experiment 1 differences between means were tested with the Student's r test. In experiment 2, radioactivity in the tissues was expressed as the mean logarithm of dpmig with a column for the SE, because of the large within treatment variation. Since the SD calculated for radioactivity in thc tissues were largely proportional to the means, it was necessary to use the logarithms of thc values for thc analysis of variance. After log transformation, the SD were more nearly equal and uncorrelated with the means (Snedecor and Cochran 1967).

339

HIDIROGLOU AND ZARKADAS

SELENIWM-SUPPLEMENTED

T

1


T

SELENIUM-SUPPLEMENTED

I

SELENIUM-DEFICIENT

SELENIUYDEFICIENT

T

4 1

2

3

4

5

6 7 8 TlME AFTER DOSING ( h )

24

FIG. 1. Distribution of radioactivity in blood plasma protein components following a single oral administration of L-["Slmethionine (5.5 ,uCi/kg BW) to sheep fed a Se-supplemented and Se-deficient diet.

Results and Discussion Experiment d Distribution o f Radioactivity into Blood Blasnza The patterns of the distribution of radiosulfur into plasma protcins of Se-deficient and Sesupplemented sheep, arc presented in Fig. 1. When 5.5 pCi/kg, BW of L-["Slmethionine (corresponding to 17.19 pmol/kg, BW) was administered intraruminally to both groups of sheep, a period of rapid increase in the level of the radiosulfur in the plasma proteins was observed. Maximum level was reached 3 h after dosing, followed by a period of progressive decline and stabilization of the radioactivity in the plasma. Downes et al. (1970) found that peak concentrations of plasma radioactivity were reached 6 h after oral administration of L-["S]methionine. Although there was a tendency for the radioactivity levels of plasma of sheep fed a Se-deficient diet to be higher than that found

T

T I

TIME AFTER DOSING d h )

FIG. 2. Distribution of radioactivity in blood plasma after intraruminal administration of L-[3S]methionine ( 5 . 5 pCi/kg BW) to sheep on a Se-supplemented and Se-deficient diet.

in Se-supplemented animals during the 24-h sampling period, these apparent differences were not found to be statistically significant (P > 0.05). The patterns of distribution of radioactivity in the ilonprotein fraction of plasma after ruminal administration of L-[:3?3]methionine arc shown in Fig. 2. An initial very rapid appearance of radioactivity in the acid-soluble componeilts of the plasma 1 h after dosing was followed by a rapid declinc, and at the 24-11 sampling the percentage acid-soluble radiosulfur was less than half (35-4096 ) that of the 1-h sample. Distribution of Radioactivity in Rurnc~nLiquor The results of the distribution of radiosulfur in the various rumen liquor fractions, summarized in Table 1, indicated that the rumen liquor from Se-supplemented wethers contained significantly ( P < 0.05) higher levels of radioactivity than the untreated animals. In the casc of the Sc-supplemented group, maximum radioactivity in the cellular and extracellular components of the supernatant of the

Sesupplemented

Sedeficient

Rumen liquor Sesupplemented

Bacteria

Sedeficient

+

2833 3448 3398 2888 1377

+ 509 + 707 + 1065 + 783

+ 1305*"

Sedeficient

Rumei~liq uorb fractions Cell free fraction Sesupplemented

"?'he data are mean values of nine determinations ? SD. bThe remainder oF the "S activity in the rumen liquor was associated wlth the ingesta fr'tstion (plant particles) NOTE:P < 0.05. * + P i0.01.

Hours after administration of ~-[3%]methionine

-

Rumen constituents, dpm!/ml of original rumen liquor

+ +

+

2436 1310 1337 f 816 1972 961 1736 f 930 1635 851

Sesupplemented

Protozoa

TABLE1. Distribution of radioactivity in the various rumen liquor fractions from Se-supplemented and Se-deficient sheep (three animals per treatment) given a single administration of L-[3%]methionine(5.5 pCi/kg BW)

+ +

1268 f 1327 1468 333 1113 341 719L364 613+102

Sedeficient


4

I .

m

\B

w

2

C] o

5

9P

2

?

r3-

"

5E

r

34 1

IIIDIROGLOU AND ZARKADAS


TABLE 2. Amino acid co~nposition'~ of rumen bacterial, cell free, and protozoal fractions for Se-supplemented and Se-deficient 5heep Rumen liquor components Bacteria Amino acid

Sesupplen~ented

Cell free extracts Sedeficient

Scsupplen~ented

Sedeficient

Protozoa Sesupplen~ented

Sedeficient

Lysine Histidine Ammonia Arginine Cysteic acid Methionine sulfone Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leueine Tyrosine Phenylalanine "The ddta are mean vaiues of nine c1ctcrmin:ations and are expressed as prnol lmg N. b+

SD.

rumen liquor was recorded 2 h after dosing, followed by a progressive decline and stabilization at 8 h. Although there was a tendency for the radioactivity levels of the bacterial and protozoal fractions of the rumen liquor of sheep fed a Se-supplemented diet to be higher than that found in Se-deficient animals during the 4- and 24-h sampling period, these apparent differences were not found to be statistically significant ( P > 0.05 ) as shown in Table 1. The results suggest that the distribution of I-[""Imethionine in rumell microbial proteins of sheep is dependent upon the selenium status of the animal. The amino acid composition of the rumen bacteria, ceIl free, and protozoal fractions are presented in Table 2, and represent the average of data from 24, 48 and 72 h of hydrolysis. There were no significant differences (P > 0.05 ) between the Se-deficient and Se-supplemented groups in the amino acid composition of their ruminal bacterial. their ceIl free, or their protozoal fractions (Table 2 ) . The evidence obtained is consistent with that reported by other authors (Leibholz 1965. 1972;

Chalupa 1972) suggesting that there is very little difference in amino acid composition of rumen bacterial protein under widely differing dietary regimes. Samples of the various rumen fractions were also oxidized as described in the Materials and Methods, and analyzed for the amounts of radioactivity in cysteic acid and methionine sulfone. As may be seen in Table 3. the specific activities of both cyst(e) ine and methionine in the 2 h rumen bacterial samples were significantly higher for the Se-supplemented wethers. Similarly, 24 h after administration of I--["~S] methionine, the amount of radiosulfur into the rumen bacterial proteins was significantIy higher in the Se-supplemented than in the nonsupplemented animals. It was also noted that the specific activities of rnethionine and cyst ( e ) ine is bacterial and protozoal protein (Se-supplemented) and of protozoal protein (Sc-deficient) increased between 2 and 24 h (Table 3 ) . However, the amino acid composition of the bacterial proteins did not appear to be influenced significantly by the selenium supplementation (Table 2 ) . A possible explanation

2d

24

+

2240 f 1085 4089 8850 1385 11499

+ 2189 + 1185 1755 2345

+ 890 + 925** +

4499 f 2286 7362 4729

9392 1 0895 2798 i 1100*

7140 992** 3980 2 1276**

+

2745*1680** 5330 1300

24

34852425'" 5765 792*$'

+

2

Se-deficient

"

2

+

5.20F4.46 7.54 IfI. 2.14

19.13 4 4.96 19.22 k 1.92

+

1.74i0.71 12.67 1.06

14.18 5 2.16 15.53 'i- 0.37

5.38k1.54 18.51 1 2.58

24

2.2640.38"* 10.49 2 2.56**

2

24 1.81i0.16 7.47 i 1.21

Se-deficient

Percent recoveries per 100 ml rumen liquorC Se-supplemented

3.4550.40 13.29 0.70

QThe data are mean values of nine determinations and are expressed as dpm per mole amino acid per milligram N. b ~ e t h i o n i n eand cyst(e)ine were determined as their oxidation products. CVallaes were expressed as percentage clist~ibutionin the rumen licluor at the indicated sampling time. asampling time in hours. SSD. NOTE: * P = 0.05. * * P = 0.01.

3. Protozoal proteins

1. Bacterial proteins 8870i1805E13940+2150 Cysteic acid Methionlne sulfone 14070 f 2095 35039 k 3570 2. Cell free fraction 25280 + 2308 9682 + 1652 Cysteic acid Methionine sulfone 10530 5 1648 6610 i 980

Amino acid

Se-supplemented

Specific activities (dpm)"

TABLE3. Percentage distribution of radioactivity in methionine and cyst(e)ine of rumen bacterial, cell free, and protozoal fractions from Se-supplemented and Se-deficient sheep following a single administration of L-[36S]methionine(5.5 pCi/kg BW)a


2

4

2

z

'a

Y


HIDIROGLOU AND ZARKADAS

343

for the differences between the results reported I 1 % of exogenous methionine was directly inin Tables 1 and 3 and those in Table 2 may corporated into ruminal bacterial proteins. be that the increased specific activity in the Coleman ( 1967) and Patton et al. ( 1970) indimicrobial protein in the supplemented sheep cated that growth of protozoa and certain reflects the differences in the rates of protein rumen bacteria is stimulated by methionine in synthesis in the rumen of Se-supplemented and vivo. This may account in part for the differences between the in vitro estimates of Nader Se-deficient animals. The isolation and purification of the bacterial and Walker (1970) and the results reported in fraction from the various samples of the rumen Table 3. On the other hand, Landis (1963) reliquor ( 100 ml) were carefully carried out ac- ported that 75-83% of the administered Lcording to the standard technique of Wright [:35S]methioninewas incorporated directly into and Hungate (1967), with the usual pre- rumen bacterial proteins of the goat. Landis cautions described by Warner ( 1962), who (1963), however, did not determine the perhave shown that there are no marked differences centage label incorporated into the methionine in microbial population from top to bottom or and cyst(e)ine of the bacterial proteins. This from place to place of the sheep's rumen pro- factor may account for the large differences bevided that not less than 100 ml of rumen tween Landis' ( 1963) estimates and the results ingesta is removed through a 1-cm internal reported in Table 3. Significantly higher levels of radioactivity diameter glass tube. It is also pertinent to note that although the overall recovery of radio- were found in the rumen cell free fraction of activity in the three rumen fractions (Table 1) Se-supplemented than in the Se-deficient accounted for about 68% of the radioactivity wethers both at 2 and 24 h after administration present in the original rumen samples, the re- of I>-["Slmethionine (Table 3 ) . These results coveries of the isolated bacterial fractions from are consistent with those reported by Champresheep to sheep in a given treatment were very don et al. (1973), who found that labeled similar. Thus, Table 3 shows that 16.75% of cyst (e) ine and methionine were present both in the radioactivity present in the original rumen the rumen microbial and extracellular fractions liquor samples of the supplemented wethers 2 h of goats following L-["S]methionine adminafter dosing, was in the isolated bacterial protein istration. Nader and Walker (1970) in their in fraction. Of this 16.75%, it was found that vitro experinlents showed that a large propor3.45% was present in cysteic acid (Table 3) tion (89% ) of the administered methionine is and the remaining 13.29% was found as degraded to sulfides in the rumen by the methionine sulfone. In the case of the Se- microorganisms and resynthesized as labeled deficient animals, the percentage of label in methionine and cyst (e) ine. Bird (1972) rethe isolated bacterial protein fraction was 12.75 ported that very little sulfide intermediates are and 9.28, at 2 and 24 h after dosing, re- found in the rumen since these intermediates spectivcly. The possibility remains that the totaI are very rapidly absorpted from the rumen in vivo distribution of L-["Slmethionine in the (Bray 1969). In the rumen protozoal fraction 2 h after adrumen bacterial protein fraction might be higher provided that part of the losses of radio- ministration of L-["S]methionine, the specific activity reported in Table 1 for the particulate activity of methionine sulfone was significantly matter is associated with microbes which might higher (P < 0.05 ) in the Se-supplemented be afixed in the solids of the ingesta. At the group than in the Se-deficient sheep. However, present time there is no definitive evidence it should be noted that there was no difference for such a possibility since neither the metabolic in specific activity of cysteic acid between the fate of the label associated with particulate two groups. Similarly, at 24 h after dosing there matter is known nor are all of the Iabeled com- was no significant difference in labeled ponents and products easily isolated and fully methionine or cyst(e)ine between the two characterized. In general, however, this data groups. Pittman and Bryant ( 1964) have shown on the isolated bacterial protein fraction is con- that methionine is required for the growth of sistent with the in vitro data obtained by Nader Racteroides rurninicola and Coleman ( 1967 ) and Walker (1970) who have estimated that found that rumen protozoa require preformed


344

CAN. J. PHYSIOL.

PHARMACOL. VOL.

TABLE4. Percentage distribution of radioactivity (log dpm/ml fresh plasma) in blood plasma following methionine ] (16 pCi/kg administration of ~ - [ h f e - ~ H BW) t o wethers fed a Se-supplemented and Se-deficient diet

T ~ B L 5L Percentage distribution of radioactivity (log dpmig freeze-dried tissue) in tissues of sheep on Se-supplemented or Se-deticient diets following a single oral administration of L-[!We-"]methionhe Tissue

Hours aftcr administration of [MP-~H]methionine

Se-supplemented Se-dcticient

SE

amino acid for their protein synthesis and that they feed extensively on bacteria (Wallis and Coleman 1967).

Experiment 2 Distribution u f Tritium in Tissues, Organs and Pkasrna after Adnzinistration of L-[Me- Wjmethionine The present experiment demonstrates that the pattern of distribution of tritium in the plasma of Se-treated and deficient animals is essentially the same (Table 4 ) , and that the levels of radioactivity of the two groups were not significantly different ( P > 0.05). Maximum radioactivity in plasma proteins for both groups was recorded between 24 and 48 h after treatment. However, it should be noted that the pattern of distribution described in Table 4 with I,-[M~-W] methionine is very different from that obtained by the use of L-["S]methionine (Fig. I ) and at the present time, there is no definite explanatioil for these differences. From the distribution of tritium in the various tissues and organs, summarized in Table 5 , it appeared that tissue radioactivity tends to be higher in sheep given a Se-supplement than in untreated ani~nals.According to Venkov and Stachev ( 1969), Se-supplementation of diets of sheep caused an increased rate of permeability of the mucous membrane of the small in-

54. 1976

Se-supplemented Se-deficient

SE

Oesophagus muscle Rumen Wall Mucosa R4uscularli~ Serosa Reticulum Omasurn Abomasum Duodenum Jejunum Caecum Colon Nasal Cartilage Taachae Ligament naet acarpal Bone marrow Muscle Heart Lung Liver Spleen Bile Adrenals Kidney-cortex Fancreas Cerebrum Hypophysi~

testine and raised tissue radioactivity concentrations in sheep. From the experimental evidence presented in Table 5, it may be concluded that the selerlium status of the sheep before administration of tritiated methionine had no significant effect on the distribution of radioactivity in any of the tissues or organs examined. These results are in agreement with the finding of Fil'Chagin ( 1965 ) that the Se status does not significantlv affect the levels of ~nethionineradioactivity in rat tissues. The highest activity levels of tritium in both groups of sheep were found in liver, kidney and pancreas and the lowest levels in the muscle and borle marrow. High levels of radioactivity were also found in the rumen mucosa of the forestomachs (Table 5 ) , which is in agreement with the work of Landis (1962). For both groups of animals the jejunum radioactivity levels were high (Table S ) , since most absorption of methionine appears to occur in this


HlDlROGLOU A N D ZARKADAS

region (Leibholz 1972; Bird and Moir 1972). In summary, it was noted that the ruminal route of administered 1,-[:IzS]rnethionine produces very similar labeling patterns in the plasma of Se-supplemented or deficient sheep, indicating that the selenium status of the sheep has no significant effect on the distribution of radiosulfur in plasma proteins. However, it may be concluded that the distribution of labeled methionine in rumen sainpIes of sheep depends on the selenium status of the sheep, with the level of radiosulfur being higher in the rumen microbial proteins of Se-suppleilaented animals than of nonsupplemented animals. It was also concluded that levels of I.-[Me-:'H]methionine radioactivity found in the blood plasma and tissues failed to reveal any significant difference ( P > 0.05) between the Se-deficient and Setreated groups.

Acknowledgment The authors are indebtcd to Dr. C. Williams of the Statistical Research Services, C.D.A., for helpful advice. The authors are also indebted to the Analytical Chemistry Services, Chemistry and Biology Research Institute, C.D.A., for the selenium, nitrogen and amino acid analyses. The skilled technica1 assistance of Miss Cari Van Es, Mr. G. Morris and Mr. E). Tute is also acknowledged. AROUAKKADA, A. R., and HOWARD, B. H. 1960. The biochen~istry of rumcn protozoa. Thc carbohydrate metabolism of Entadiniunz. Biochem. J. 76, 445-45 1. BIRD, P. R. 1972. Sulphur metabolism and excretion studies in ruminants. V. Ruminal desulphuration of methionine and cyst(e)ine. Aust. J. Biol. Sci. 25, 185-193. BIRD.P. R., and MOIR,R. J. 1972. Sulphur metabolism and excretion studies in ruminants. Methionine degradation and utilization in sheep whcn infused into the rumen or abomasum. Aust. J. Biol. Sci. 25, 835-848. BRAY,A. C. 1969. Sulphur metabolism in sheep. I. Preliminary investigations on the movement of sulphur in the sheep's body. Aust. J. Agr. Kes. 20, 725-737. CAIL'TONI, G . L. 1953. ,5'-Adcnosylmethionine; a new intermediate formed enzymatically from L.mcthionine and adenosinetriphosphate. J. Biol. Chem. 204,403-416. CHALUPA, W. 1972. Metabolic aspects of non protein nitrogcn utilization in ruminant animals. Fed. Proc. 31,1152-1 164.

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CHAMPREDON, C., PION, R.. and PRUGNAUD, J. 1973. Etude du metabolisme de la mcthionine clans le rumen de la chkvre. Ann. Biol. Anim. Hiochcm. Biophys. 13,774-775. COHFK. G. N., and Cowrs, D. H. 1957. Replacement total de la methionine par la selenomethionine dans les protkines d'Esclrericliicr c d i . Compt. Rend. Acad. Sci. 244,680-683. COLFMAN C;., S. 1967. The metabolism of frec amino acids by washed suspensions of the rumcn ciliate Entodinilim cozitlatr4rn. J. Gcn. Microbial. 47, 43 3-447. Dowhr s, A. M.. REIS,P. J., SIIARRY. L. F., and TUNKS, D. A. 1970. Evaluation of modified ["SJ-Lmethionine and [""S] casein preparations as supplements for sheep. Br. J. Nutr. 24, 1083-1089. FII

The metabolism of methionine in silage-fed sheep.

The effects of selenium depletion and repletion on the metabolism of thyroid hormones in the rat.

The effects of hypoxia on (methionine) enkephalin peptide and catecholamine release in fetal sheep.

Effects of selenium deficiency on vitamin E metabolism in rats.

Effect of cystine on the metabolism on methionine in rats.

Methionine metabolism in BHK cells: the regulation of methionine adenosyltransferase.

Some effects of sulphur intake on molybdenum metabolism in sheep.

Effects of high dietary lead on the metabolism of intravenously dosed selenium-75 in dairy calves.

Interactive effects of selenium, methionine, and dietary protein on survival, growth, and physiology in mallard ducklings.

In vitro metabolism of selenite in sheep blood: factors controlling the distribution of selenium and the labelling of plasma protein.

Effects of selenium and retinoic acid on the metabolism of N-acetylaminofluorene and N-hydroxyacetylamino-fluorene.

Effects of sex hormones and of pregnancy on the selenium metabolism.

The effect of copper excess on iron metabolism in sheep.

Selenium residues in sheep meat.

Tissue nonautonomous effects of fat body methionine metabolism on imaginal disc repair in Drosophila.

Effects of hypothyroidism and thyroxine substitution on the metabolism of L-methionine, L-cystathionine and taurine in developing rat brain.

Effects of lactation on the regulation of hepatic metabolism in the rat and sheep: adrenergic receptors and cyclic AMP responses.

Effects of copper and selenium supplementation on performance and lipid metabolism in confined brangus bulls.

Metabolism of propionate in the tissues of the sheep gut.

Effect of selenium supplementation on thyroid hormone metabolism in an iodine and selenium deficient population.

Disturbed methionine metabolism in pancreatitis.

Antagonistic effects of selenium on cadmium-induced apoptosis by restoring the mitochondrial dynamic equilibrium and energy metabolism in chicken spleens.

vitamin B-12-methionine-deficient diet.

Effects of Perilla ketone on the in situ sheep lung.