multiple forms of lactate dehydrogenase and

MULTIPLE FORMS OF LACTATE DEHYDROGENASE AND ASPARTATE AMINOTRmSFERASE IN HERRING QCLUBEA HARENGUS HARENGUS L.)

Can. J. Biochem. Downloaded from www.nrcresearchpress.com by University of Alberta on 03/27/15 For personal use only.

PAULH. OBEXSE,THERESA &%.ALLEN,AND TEDC. LEUNG Fisheries Research Board of Caaada, Halifccx Laboratory, Halifax, Nova Scotia Received April 22, 1966

Abstract The distribution of isoenzymes of lactate dehydrogenase (LDH) and aspartate aminotransferase (Apa'H')in the tissues of 189 herrin (Qupea h a w n g u s ktareagus L.) were examined. Starch-gel electrophoresis of the L ~ isoenzymes H of the herring revealed the presence of two hybrid forms representing mutant alleles a t the B locus. These mutants gave rise to two genotypes, BBQnd BBrP,whose LDH staining patterns revealed a binomial distribution of the tetramer combinations formed from a free and random association of the A, B, and B', and the A, B, and B" monomers respectively. A hybrid form of soluble AAT was found. Its electrophoretic pattern showed a 1:2:1 binomial distribution of bands. I t is postulated that these bands represent ,\AT dimers formed from normal S and mutant S P monomers of a heterozygous SS' genotype. The normal homozygous SS genotype showed only one band of activity. The normal levels of LDH and I U T activity in plasma and in heart and skeletal nauscles were determined. During frozen storage LDH-5 activity gradually disappeared, while LDH-1 activity changed least; LBH-1 was also most stable a t higher temperatures. Frozen storage rapidly destroyed AAT activity.

AppeIla and Marker$ (1) demonstrated that the enzyme lactate delaydrsgenase (LDH)l is a tetramer, which was subsequentIy shown to consist of two different kinds sf monomers designated "A9' and 46Bv' by hIarkert (2). The 66A0and 6 6 B pmonomers p are urnder control of separate genetic loci and the association of these two monomers into tetramers gives rise to the five isoenzymes of LDH in mammalian tissues. Sub-banding of the five isoenzymes is frequently observed, and in a recent review Markert (3) suggested three explanations: (i) the existence of additional monomers under the control of further genetic loci (such as the "C" subunit found in sperm by Blanco and Zinkham (4)) ; (ii) permutations sf the monomers within the five major bands, (e.g. a t e t r m e r with a monomer sequence ABAB might differ in mobility from a tetramer of the saille subunit composition but with a different monomer sequence ABBA) ; and (iii) the existence of mutant alleles a t any 0%the existing loci. The LDH patterins of fish differ considerably from the mammalian pattern sf five bands. Ibfarkert and Faulhaber (5) studied 30 species and found fish with I, 2, 3, or 5 LDH isoenzymes. They suggest that factors such as subunit charge and molecular configuration afFect the combination of fish LDW monomers more than the mammalian monomers. Thus some subunit associations are preferred and most fish fail t o show all five major LDH isoenzyme bands. IL-Lactatei NAD oxidorductase (EC 1.1.1.27). Canadian Journal af Bioclhemistry. VoIlame 44 (1966)


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CANADIAN JOURN-SL OF BIOCHEPrTISTRU. VOL. 4.1, 1960

Though the majority of fish possess two mononiers, hlarkert and Faullaaber postulate that the fluke (Paraickztlz,ys dentalus) and related species represent a group which developed only one monomer under the control of a single gene, and consequently this group possesses only one LDH isoenzyme. The enzq7me aspartate aminotransferase (A%hAT)%ccursas both a bound mitochondria1 and a soluble enzyme. Various workers (Boyd (61, Hook (%), M'orino ef a&.(81, and Martinez-Carrion et al. (9)) have shown that these two forms possess different electrophoretic n~obilities,molecular \veights, solalbilities, and pH optin~a.WIartinez-Carrion et al. (9) have shown that the soluble form of the pig-heart enzyme can be separated into three active fractions by starch-gel electrophoresis. Block et al. (18) demonstrated the presence of five isoenzymes in human serum and Decker and Rau (11) found up to four isoenzj7mes of AAT in heart muscle extracts of various rnalnnaalian species. Recently Polyanovsky (12, 13) studied a purified preparation sf soluble AAT from pig heart and demonstrated that the enzyme exists as a dimer in concentrated solution, but dissociated into an active monomer in dilute solution. At higher pH values he was able to bring about a reversible dissociation sf the dimer by treatment with succinic anhydride. In the present investigation, two catches of herring (Clupea harengus Bzarengus L.) were examined for LDEI and AA4T isoenzyn~esby starch-gel electrophoresis. In addition, the enzg7melevels were determined quantitatively by colorimetric procedures. The effects upon the enzymes of high salt concentrations, freezing, heating, and storage were determined. Attempts were made to dissociate the LDH and AAT enzymes into their constituent monomers, follo~veefby recoml-,ination into new and active polymers.

Methods Herring were collected in the Atlantic coastal waters near Halifax, Nova Scotia. Separate catches of 15 and 114 herring respectively were held alive in recirculating seawater tanks. Before use the fish were anesthetized in a so1utio1-aof tricaine methanesulfonate3 1:20,1000 in sea~vater.The length, sex, weight, and coladition of each fish were recorded. Blood was obtained from each fish either through cardiac paarlcture or from the dorsal aorta. Extracts sf white skeletal muscle were prepared from all the herring in catch I (15 fish) by blending one part nauscle with two parts distilled water for 1 minute a t 0 ' C in a Servall Omnimixer. Heart muscle extracts evere sin-ailarly prepared from all the fish in catch I. The extracts were centrifuged a t 12,0010 g for 30 minutes and the supernatants were used directly for electrophoresis. The second catch of herring was screened for the presence of hybrid AAT patterns by examining the plasma of each fish. Extracts of skeletal white muscle, skeletal red muscle, smooth muscle, heart muscle, brain, eye, t-Aspartatez2-oxogluhrate aminotransferase (EC 2.6.1.1). Went Chetnical Co., Va~leouver,British Colcarnbia.

BBEXSE ET A%.: ENZYMES IN WERRIKG

1321


gonad, kidney, liver, and spleen were then made on a representative sampling of the herring in catch 11. Preliminary work in this laboratory demorlstrated that starch-gel electrophoresis gave consistently better results in the electrophoresis of fish tissue extracts than did polyacrylarnide gel s r cellulose-acetate strip electrophoresis. Markert and FauEhaber (5) similarly reported that starch-gel electrophoresis produced a better resolution sf crude hsmogenates. The electrophoresis ~f the hearing samples, therefore, was carried out by the vertical starch-gel procedure sf Smithies (14), with a modified Aronsson and Gron1va11 (15) buffer .cvhich consisted of (grams per liter) tris(hydruxymethyl)aminoirlethane (Tris) 15.1, ethylenediaaninetetracetic acid (EUTA) 1.5, and boric acid 1.15. The pH was adjusted to 9.0. The running ~ i m ein most cases mas 6 hours, made at a constant voltage of 400 and a current of 40-50 mA. Some samples were run for 18 hours a t 300 V a t a current of 20-25 nlA and resulted in a clearer separatioil of LDH isoenzyrnes, especially when multiple sub-banding occurred. Quantitative LDH levels were determined according to the colorimetric method of Cabaud and LVroblewski (16) described in Sigma Technical Bulletin No. 500.4A Coleman junior spectrophotometer was used to measure the optical density of the pyruvic acid hydrazone a t 525 n ~ p The . activity is reported in Berger-Broida LBH units per milliliter ; one Berger-Broida unit is the amount of enzyme which will reduce 4.8 X %dBe4 pmole of pyruvate per minute a t 25 "C. After electrsphoresis, the L B H bands were made visible by incubating one half of the sliced gel in a staining mixture which was essentially that described by Dewey and Conklin (171, and consisted of Tris-HCl buffer, 0.2 Jf, pH 8.0, 12.5 ml; sodium L-lactate, 2.8 A[, pH 7.0, 1.5 m1; p-nitro-blue tetrazslium salt (NBT), 0.5 rng/~ml, 37.5 ml; phenazine methosulfate (PMS), 0.2C;I,, 0.6 ml; nicotinamide-adenine dinucleotide (NAD), 35.0 m1. The gels were developed from 3 to 16 hours depending upon the LDH activity. Controls were run by incubating gel halves in staining mixtures lacking either sodium L-lactate, PMS, NAD, or both NAD and PATS. A small amount of activity attributed to nsn-specific or "nothing' dehydrogenases was detected in the control gels, but in n s case did it approach that of the L B H bands in the complete system. The AAT activity nTasdetected on the starch gels by an adaptation of the procedures sf Babssn et al. (18) and Decker and Rau (11). After electrophoresis, the top half of each sliced gel was i~scubatedin a mixture of 25 ml of the buffered substrate solution of Babson et al. (18) and 25 ml of diazonium salt solution. The buffered substrate solution contained 146.1 mg a-ketoglutaric acid, 532.4 mg L-aspartic acid, 5.68 g NaHzP04, 2.8 g pslyvinylpyrrolidone (PVP), 0.2 g EBTA, dissolved in water to make 200 ml of solution and adjusted to pH 7.4. The diazsnium salt solution contained 5 mg 6-benzamido4-methoxy-m-toluidine diazonium chloride per milliliter. The gels were incubated for 4-1 hour, then washed in distilled water, and preserved in 5% acetic 'Sigma Chemical Co., St. Louis, Missouri.


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CANADIAN JOURNAL O F BIOCIIEMPSTRY. VOL. 44. 1966

acid solution. Pictures were taken irnn~ediately,as the bands tended to fade s n storage. -4spartate aminotransferase controls were run by incubating the other half of each gel in the same staining solutions as above but without a-ketoglutaric and L-aspartic acids. All control gels were negative. The AAT levels were determined according to the method of Babson et a&. (18) using the 'H'rans-Ac reagent k i t . V h e Trans-Ac unit is defined as the amount 0%enzyme that \%-illform l p m ~ I eof oxalacetic acid per minute per liter of serum under tlne specified conditions (pH 7.40, 25 'C).

Results and Discussion Lactate Behydrogenase Herring was found to have five bands sf LDH activity. They were designated LDl-I-I to LDH-5 in order of decreasing mobility towards the anode. There were three major bands of activity, LDH-1, LDH-3, and LDH-5, shown in Fig. 1, Nos. 4, 5, and 6. Weak bands of activity were present a t the LDH-2 position in heart and skeletal muscles, and a t LBH-4 in heart muscle, but these bands were frequently absent. This is in agreement with the work of Markert and Faulhaber (51, who showed that herring possesses five LDH isoenzymes, with LBH-2 and LDH-4 difficult to demonstrate. The conventional subunit formulations for LDH-1, LDH-3, and LDH-5 are B2A2?and Ag respectively. The subunit formulations for weak bands LDH-2 and LDH-4 are AB3 and I t is possible that the subunits assemble preferentially in hoinodimeric pairs AA or BB, rather than hetersdirneric pairs AB, before assembling into tetramers. The preferential hsmodirneric associatioas would explain the strength of the LDH-1, EDH-3, and LDH-5 bands, and the relative m-eakness of the LDH-2 and LDW-4 bands which contain heterodimers. Two hybrid patterns tarere found with multiple sub-banding. The first occurred wit11 a frequency of 6 in 7'5 in catch I, and 17' in 114 in catch 11 (Fig. 1, Nos. 1, 2, and 3). The second hybrid pattern occurred only oilce in catch 11 (Fig. 2, No. 3). This type of sub-banding was attributed by Rlarkert (5) in his studies of whiting (MerJuccius bilinearis) to mutant alleles (B' and BP') a t the B loci. The sub-banding observed in the herring seems to be an example of ttaro different mutations occurring a t the B locus, which would result in six different genotypes of herring with the postulated patterns shown in Fig. 3. A fourth pattern tvas evident in some of the herring LDH patterns. In this type, weak bands of activity appeared between LBH-1 and LDH-2, and between LDH-2 and LDH-3. These bands were interpreted as representing permutations of the basic tetrameters. Dilution of the concentrated sample did not result in any change in tlne pattern (Fig. 4, Nos. 1-41. "repared Ontario.

reagents are avaiHable in kit %ormfrom Warmer-Ckilcstt Laboratories, Toronto,


(LDH) activity after starch-gel electrophoresis for FIG.I. Herring lactate dehydroge~~ase Br3 hours a t 300 V. Slots 1, 2, arid 3, heart mrlscle extract, plasma, and white skeletal muscle extract respectively from BB' herring. Slots 4, 5, and 6, heart nluscle extract, plasma, and white skeletal nluscle extract respectively from I3R herring. FIG. 2. lieart rntlscle lactate dehydrogenase (LDH) patterns of the different herring genotypes after 6 hours' electrophoresis a t 400 V. 1 , RE' herring; 2, HI3 herring; 3, BR" herring. Skeletal muscle B,I>H patterlis of these herring showed the i l c barid, which is often absent in heart nntascle preparationas.

QDENSE ET Ak.: ENZYMES IN HERRING


LDH

GENOTYPES

B LDH-I Q . B 4

~

B

;

~3~~3' D B 4

FIG. 3. Postulated lactate dehydrogenase patterns for the six possible herring genotypes resulting from the r~ormaland two mutant alleles.

The observed distribution of the different genotypes in catches 1 and IH, the allele frequencies, and the calculated distributions of the unobserved but postulated genotypes B'B', B'B", and B"BPEare presented in Table 1. These TABLE Y: Genotypes of herring lactate dehydrogenase (LDH)

--

LDH genotype distribution (observed)

BB BR' RB" Total Catch1 Catch I1

67 96

8 17

0 1

75

114

.

Allele frequency

B

B'

B"

0.055 0 0 . I95 0.080 0.00.5

0.945

Distribution of other 1,DH genotypes (calculated) BIB!

BPB~P

BPIRII

0 0 f in 330 l in 159 l in 1250 l in 4080

latter distributions were calculated by applying the Hardy-Weinberg law t o the observed values for the allele frequencies. Of the unobserved genotypes, only the BIBP type is apt to be found in a sampling of the size used in this study. The number of sub-bands and their intensity of staining in the case of the BB' and BBPPgenotypes reflect a binomial distribution corresponding to random association into tetramers of naonorners A, B, and B', and A, B, and B" respectively (LDH-3, I:2: P and LDH-I, '%:4:6:4:I).


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CANADIAN JOURNAL OF BIOCHBMZSTRPr. VOE. 44, 1966

Herring LDH exhibited the typical mammalian pattern, in which skeletal muscle has higher concentrations of LDH-5 and heart rrnuscle has higher concenntrations of LDH-3 and LDW-1 isoenzyrnes (Fig. 5, Nos. 1 and 3). The isoenzyme distribution was similar for all tissues (Fig. 5 ) and whenever multiple sub-banding occurred it was present in all tissues. hIuscle extracts prepared a t 0 'C were incubated a t 25", 37", and 57 "C for 3 hours and the LDH activities were determined quantitatively. Herring LDH activity was not affected by incubation a t temperatures up to 37 OC, but a t 57' C nearly all the activity was lost (Table 11). Starch-gel electrophoTABLE I1 Lackite dehydrogenase (LDH) levels in plasma and tissue extracts (units/mlj Incubation temperature Tissue Heart muscle extract Skeletal muscle extract PHasma

0 OC

25 "C

37 OC

57 "6

53,000 8'7,CBW 28,000

44,000 88,000 23,000

33,000 27,000 12,008

5,300 217' 833

resis revealed that tfae s~nallamount sf activity remaining after incubation at 57 "C was present in the LDH-1 band. During storage a t - 15 "C, LDH-5 activity disappeared completely within 2 months, EDH-3 activity declined more slsevly, and LDH-1 was the most stable. The activity found in herring muscle ~ v a sseveral-fold higher than the corresponding r~~ailalrnalian levels. The method of 'Tarkert (3) was used to attempt fully to dissociate herring LDH into its monomer subunits, followed by free re-association into new conabinatiorns. Tlae expected binomial distribution (1 :4: 6:4: 1) of the five reassembled EDH iscseazyrnes did not occur (Fig. 6). Instead there was no change in the BB' pattern after treatn~emat,whereas the BB pattern showed an increase in sub-bands bet\\-eena LBH-1 and LDH-2, and between LDH-2 and LDH-3, and a slight increase in LDH-2. Thus it is concluded that EDH-2 and LDH-4 combinations are a t least partially 'forbidden' and the au b-handing which did occur uTasbrought about by permutations of the tetramers. A comparison was made of the t~vohalves sf a gel, one half stained with anxido black for protein, the other stained for L1)I-H activity. Three different LDH types were selected and, as shown in Fig. 7, the species-specific protein patterns stained with arnido black did not reflect a n y of the differences found in the corresponding LDH patterns.

Asfiartde Aminotransferase Herring AAT showed patterns similar to that found by Boyd (61, Hook (a), and faqorina et al. (8). The n~itochsndrialLALA'$ migrated slowly towards t h e cathode, and the soluble AAT migrated mare rapidly tom-ads the anode (Fig. 8, NOS.1,2, and 3). Four herring out of 75 in catch I showed a second type of


FIG. 4. 1lilutio~1effect on white skeletal rnuscle lactate dehydrogenase (EDH) pattern. RB genotype showing sub-banding attributed to permutations of 1,DH-I and I,ll)H-3 tetramers. Sluts 1 4 , dilutions of muscle extract with 2, 4, 8, and I6 parts respectively of distilled water. FIG. 5 . Ilistribution of lactate dehydrogenase (LDH) i11 BH genotype herring tissues: I, white skeletal muscle; 2, red skeletal muscle; 3, heart muscle; 4, intestine; 5, liver; 6, kidney; 7, spleen; 8, ovary; 9, eye; 10, brain.


FIG. 6 . Effect of dissociation and random associatioil of lactate dehydrogenase (1,DH) isoenzymes of a BB herring: 1, 2, and 3, i~ornidlplasrma, heart muscle, and skeletal muscle; 4, 5, 6, heart muscle, skeletal muscle, and plasma respectively, after freeze-thaw treatment in 1.0 ik2 NaCi :at p%i 7.0 followed by dialysis a t 0 O C for 18 hsrnrs against distilled water. Frc. 7. Coanparisoii of ainido black stairled protein patterns with the corresporadiilg lactate dehydroge~lase(LDH) stained gels: 1, 2, and 3, atnido black stained protein patterns; 4, 5, arad 6, I,I)H patterns corresponding to 1, 2, and 3. Germtypes RB, RB with pernlutatloil sub-banding, and BR'.


FIG.8. Herring aspartate arnir1otr;ansferase (AA'l') activity after starch-gel electrophoresis for 6 hours a t 400 V. Slots 1, 2, and 3, white skeletal muscle, heart ~nuscle,and plasma respectively of SS genotype herring; slots 4, 5, and 6, white skeletal muscle, heart muscle, and plasma of SS' genotype herring.


FIG. 9. Distribution of aspartate aminotransferase (XA?') activity in herring tissues: 1, brain; 2, eye; 3, ovalq.; 4, spleen; 5, kidney; 6, liver; 7, intestine; 8, heart muscle; '3, red skeletal muscle; 10, white skeletal muscle.

BBENSE BT AL.: ENZYMES IN PPERWZh-G

1325


pattern in which the soluble fraction was split into three sub-bands. The nniddHe sub-band stained twice as intensely as the other ttvc~(Fig. 8, Nos. 4, 5$ and 6). This pattern occurrecl in only 1 herring out of 114 in catch 11. These sub-bands can be interpreted as resulting from a mutant allele for the soluble S type AAT enzyme. Three genotypes of herring would then be possible, as illustrated in Fig. PO. The observed and calculated distributions sf AAT genotypes and allele frequencies are presented in Table 111. AAT

GENOTYPES

FIG. 10. Postulated patterns for the three possible herring aspartate arninotrallsferase genotypes resulting from the cornhinatiai~of normal S and mutant S' alleles.

Genotypes of herring aspartate aininotr;~i~sferase

(Am) AAT genotype distribution (obswed) SS

SS'

Allele frequency Total -

Catch 1 Catch I1

71 113

4 1

-

75 I14

-

--

S

0.975 8.995

S9S9

SF - --

-

Distribution of other AAT genotype (calculated)

-

0.025 0.005

1 in 1600 1 in 4800

If ALqT is a dixner, as suggested by Polyanovsky (12, 131, then the S4 genotype pattern avsuld have one band between the single bands of the SS and Sf%' types with the bands showing a binomial distribution (1: 2: 1). This agrees with our sbssrvatio~asand indicates that the solarlale AAT enzyme of a herring is a dirner. The AAT enzyme was stable when incubated a t 25" and 3%'C, but all activity was lost after incubation at 57 "C. The AAT activity decreased rapidly in frozen storage a t - 15 "C (Table HI'). White skeletal, red skeletal, blsparhte aminotransferase (AAT) levels iai plasma and tissue extracts (ranits/liter)

Heart muscle extract Skeletal muscle extract I'lasana

Fresh sample

Frozen sample after 2 xnonths9 storage a t - 15 "@

100,080 800,000 13,250

1,750 5,750 P ,475

-


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CANADIAN JOURNAL O F BHOCHEhfISTR$7. VOL. 44, 1966

smooth, and heart muscles, brain, eye, liver, spleen, kidney, and gonad tissue were examined for AAT activity (Fig, 9, Nos. 1-10). Either all the tissues showed a single band of soluble AAT activity, conforming t o the SS genotype, or they all displayed the nzultipIe banding of the SS' type. Sub-banding in the soluble fraction was not acconlpanied by sub-banding in the mitochondria1 fraction. This was taken as further evidence that the mitochondrial and soluble forms of the enzyme are under separate genetic control. The methods of both Alarkert (3) and Polyanovsky (13) were used in a n attempt t o dissociate the herring AAT enzyaaaes into their '%I" and ""S" n~onsr-rlers,follo\ved hy a recon~lsination of the monomers to form a new hybrid ""ShI" band with a postulated position interilldiate betxveela the mitochondrial and soluble AAT bands. 'khese nlethsds were without effect. Possibly t h e dimer cannot be forn-maed,or is excluded by the preferential association of S monomers into SS dimers. Ixadividual fish demonstrated the same genotype pattern in all tissues whether hybrid or nornlal, LIIH enzyme or AAT enzyme. I t is evident t h a t both LDH and AA'T tissue enzyme patterns are reflections of direct genetic control and are unaffected hg- physiological or environmental factors smelt-a as the stage sf maturity, sex, o r rautritional state of the individual. These Baybrid forms should therefore prove useful as markers for the characterizationn sf herring popuHatisnns.

References 1. E. APPELEAand C. k. MARKERT.Biochem. Biophys. Res. Commun. 6, 1'71 (1961). 2. C. k. MARKERT. 6?8 Hereditary, developmental armd irn~nunologicaspects s f k i d ~ ~ e y disease. Edited by J. Metcoff. Northwestern Univ. Press, Evanston, Illinois. 1962. p. 54. 3. C. L. ~ ~ A R M E R T .In The Harvey kectures, .%ries 59. Academic Press, Inc., New York. 196%.p. 187. 4. A. BLAKCO and iV. H. ZPNKHAM. Science, 139, 601 (1963). and I. FAULHABER. J. Exptl. Zool. 159, 310 (1965). 5. C. E. MARKERT 6. J. iV. BOYD. Biocheln. j.81,434 (1961). 7. R. H. I-1009. Dissertation *\bstr. 23,419 (1962). 8. Y.~ I O K I N B H., ITOH,and H. ~VADR.Biochem. Riopkys. Hies. Cornlnun. 13, 348 (1963). F. RPVA,C . TURANO, and P. FASEELA. Riochem. Biophys. Ibes. 9. M. ~IARTINEZ-CARRIOK, Comrxiun. 28, 206 (P!)G). D. BLOCK,1%. CAKMICHAEL, and C . E. JAGMSCBW. Proc. Soc. Exptl. Biol, &led. 115, 10. 941 (1064). 11. L. E. DECKERand E. M. RAW. I'roc. Soc. Exptl. Biol. Med. 882, 144 (1963). 12. 8. BA. I P o ~and ~V. I.~ IVAXOV. ~ ~ Biokhimiya, ~ s ~29, 728 ~ (1964). 13. 0. k. POL~AKCIVSKY. Biochem. Biophys. Res. Cornrnun. 69, 364 (1905). 14. 0. SMITHIES.Biochem. J. 61, 629 (1955). atad A. GRBNWAEL.Scand. J . Cli11. 1,ab. Invest. 9,338 (1057). 15. T. AR(?NSSCPN 16. P. G. C A B A ~an$ D F. ~%'ROBEEWSKP. ir$m. J. @linePathol. 30, 234 (1958). 17. A/I. AT. DEWEYand 4. I,. COKREIM.Proc. Soc. Exptl. BisH. Med. 105, 442 (1960). 18. A. k.WABSON, P. 0. SHAPIIZO, P. A. R. N'ILLIAMS, and GoE. PHIILLIPS.Cli11. Chirn. .%eta, 7, 109 (1962).