The compositions of nitrogen pools of ox liver, bladder bile, kidney and lung were analyzed with an especial bearing on their minor components, and some distinctive features of these tissues were described. DCEC and CMC were found in ox liver and kidney. Liver was low in free arginine and lysine, but high in ornithine, ethanolamine,
and glutathione. Glycine was only a predominant amino acid in ox bile. All amino acids were contained moderately in kidney, but glutathione content was low.
The concentrations of arginine and lysine were relatively high in lung.</p

Azumi, Tsukasa

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Studies on nitrogen pool of animal tissues. 3. Ox liver and bile. IV. Ox kidney and lung

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Acta Medica OkayamaVolume 21, Issue 6 1967 Article 5DECEMBER 1967Studies on nitrogen pool of animal tissues. 3.Ox liver and bile. IV. Ox kidney and lungTsukasa Azumi∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Studies on nitrogen pool of animal tissues. 3.Ox liver and bile. IV. Ox kidney and lung∗Tsukasa AzumiAbstractThe compositions of nitrogen pools of ox liver, bladder bile, kidney and lung were analyzedwith an especial bearing on their minor components, and some distinctive features of these tissueswere described. DCEC and CMCwere found in ox liver and kidney. Liver was low in free arginineand lysine, but high in ornithine, ethanolamine, and glutathione. Glycine was only a predominantamino acid in ox bile. All amino acids were contained moderately in kidney, but glutathionecontent was low. The concentrations of arginine and lysine were relatively high in lung.∗PMID: 4236511 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Moo. Okayama 21, 321-326 (1967)STUDIES ON NITROGEN POOL OF ANIMAL TISSUESIII. OX LIVER AND BILEIV. OX KIDNEY AND LUNGTsukasa AzUMIDepartment of Biochemistry, Okayama University Medical SchoolOkayama, Japan (Director: Prof. S. Mizuhara)Received for publication, October 20, 1967Continued from the first paper of this series\ the nitrogen pools of ox liver,bile, kidney, and lung were analyzed with a special emphasis on their minorcomponents.Among cysteine conjugates2 which have been found in urine, S-(1.2-dicarbo-xyethyi)-L-cysteine (DCEC)S and S-(carboxymethyl)-L-cysteine (CMC)4 werefound in liver and kidney.MATERIALS AND METHODSOx liver (500 g), bile (500 ml), kidney (500 g), and lung (200 g) wereobtained fresh from a slaughter house.The ampholyte fractions of liver, kidney, and lung were prepared by thesame method as described in the previous paperl •The ampholyte fractions of bile were prepared as follows. Five hundred mlof bladder bile was made weakly acidic with acetic acid and filtered. The filtratewas treated in a separatory funnel three times with the same volume of etherand then once with petroleum ether. The water layer was transferred on acolumn containing Diaon SK·1 (500 ml, H-form of strong cation exchanger, mesh100), and the column was washed with 2 liters of water, 3 liters of 95 %ethanol, and 3 liters of water in that order. During the ethanol washing, somewhite solid appeared in the effluent. The solid may be steroids and bile salts.The Diaion SK-1 column was finally eluted with 3 liters of 2N-NHs andthe ammonia eluate was evaporated to dryness under reduced pressure. Theresidue was dissolved in a proper volume of water and filtered. The ampholytesin the filtrate was divided into three Fractions (I, II, and III) on an AmberliteCG-4B column as described in the previous paper\ Fraction I contains mainlybasic and neutral ampholytes, Fraction II acidic ampholytes, and Fraction IIIstrong acidic ampholytes. Each fraction was analyzed on an automatic aminoacid analyzer before and after hydrolysis.3211Azumi: Studies on nitrogen pool of animal tissues. 3. Ox liver and bileProduced by The Berkeley Electronic Press, 1967322 T. AZUMIIf necessary, the hydrolysates of these fractions were again fractionated onAmberlite CG·4B column.The Fraction IV of the bladder bile was prepared as follows. The effluentand washings (water and 95 % alcohol) from the above Diaion SK·1 columnwere combined and made weakly alkaline with dilute sodium hydroxide solution.The alkaline solution was concentrated to about one liter and filtered. Solidsodium hydroxide was added to the filtrate to make the final concentration about10 %, and the solution was boiled with a 'reflux condenser on a sand bath for24 hours. The hydrolysate was acidified with concentrated hydrochloric acid ina sparatory funnel and the precipitate appeared was removed with ethyl etherby occasionally shaking. The water layer was evaporated to dryness repeatedlyto remove excess hydrochloric acid. The amino acids in the dried residue wascollected by using Diaion SK-1 column. This is Fraction IV of bladder bilewhich contains N-covered ampholytes. The method of preparation of FractionIV of the other tissues was described in the previous paper l .RESULTSThe amounts of usual amino acids and glutathione were summarized inTable 1. The values were expressed in fl. moles per 100 g of wet weight or per100;ml of bile. The values in parentheses were those obtained after hydrolysis.I. Ox LiverLiver contained S·(1.2·dicarboxyethyl) glutathione (DCEG)5 and S·(1.2·dicar·boxyethyl)-L-cystein (DCEC)3 in Fraction III. The total amount (0.39 p. moles/100 g) was calculated as DCEC after hydrolysis of Fraction III. The hydrolysateof Fraction III was fractionated on Amberlite CG·4B column with 2M-acetic acidand then 2N-hydrochloric acid. DCEC was found only in the 2N-HCI eluate.When the hydrolysate of Fraction II was fractionated on a long columncontaining Diaion SK-l with 0.5N-hydrochloric acid by using a fraction collector,a small amount of S-(carboxymethyl)-L-cysteine (CMC)4 appeared after asparticacid. CMC was identified on paper electrophoresis and on amino acid analyzerby comparing with authentic CMC4.The other distinctive featur,=s of the nitrogen pool of ox liver were asfollows. A negligible amount of arginine was found before hydrolysis and noremarkable increase of arginine was observed after hydrolysis. Ornithine con-tent, however, was fairly high. This might be due to a strong arginase activityof liver. Citrulline was not detected either. Free lysine was not detected, but itappeared after hydrolysis. Histidine content was doubled after hydrolysis. Twounknown peptides were seen around carnosine and just after ammonia peak, the2Acta Medica Okayama, Vol. 21 [1967], Iss. 6, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol21/iss6/5Nitrogen Pool of Animal TissuesTable 1323Aspartic AcidThreonineSerineGlutamic AcidProlineGlycineAlanineHalf CystineValineMethionineIsoleucineLeucineTyrosinePhenylalanineHistidineLysineArginineOrnithine'Y-Aminobutyric AcidEthanolamineGlutathioneLiver103.81 (100.18)15.93 ( 49.63)125.94 ( 53.00)387.60 (718.05)44.32 ( 29.92)361.58 (779.20)211.68 (239.20)46.40 (242.40)35.04 ( 52.80)+ ( 6.40)22.08 ( 37.60)36.40 ( 36.96)12.80 ( 9.92)11.68 ( 9.60)36.16 ( 65.28)- (41.44)+ ( 7. 2)52.64 ( 58.56)+ ( + )157.19 (104.32)193.42 ( - )Bile1.56 ( 2.97)0.74 ( 1.72)1.84 ( 2.46)10.32 (15.20)1.10 ( 2.93)52.15 (79.37)4.49 ( 6.99)0.61 ( 0.76)1.23 ( 2.74)0.73 ( 0.34)0.38 ( 1.27)1.61 ( 1.93)0.41 ( 0.58)0.59 ( 0.86)0.25 ( 4.98)0.46 ( 1. 75)+ (0.75)0.38 ( 0.46)- ( - )2.63 ( 3.54)- ( - )Kidney89.75 (157.48)23.07 ( 67.21)32.92 ( 68.80)361.50 (807.12)29.88 (130.86)320.29 (735.42)78.69 (182.66)22.20 (140.74)20.40 (39.44)+ (14.13)13.14 ( 34.18)25.92 ( 60.16)10.98 ( 28.04)10.74 ( 29.04)12.54 (129.28)18.73 (100.48)10.97 ( 37.36)10.38 ( 39.52)4.64 ( 14.96)31.48 (103.38)5.02( -)Lung84.82 (108.63)3.72 ( 13.84)32.31 ( 28.84)311.03 (463.11)27.97 ( 43.78)510.08 (739.65)96.47 (103.63)37.31 (104.03)34.42 ( 36.00)+ ( 8.16)1~.61 ( 19.35)36.60 ( 40.80)13.51 ( 12.40)16.68 ( 17.50)10.09 ( 14.87)30.14 ( 54.00)20.07 ( 23.87)+ ( 5.5)- ( + )47.87 ( 84.00)29.43 ( - )latter might be a lysine containing peptide. After hydrolysis of Fraction I, 13-alanine increased from 30.43/1. moies/IOO g to 41.31/1. moles/lOa g. y·Amino-butyric acid was negligible before and after hydrolysis. The amount of carno-sine-like peak (about 21/1. moles/lOa gm) exceeded the increment of 13·alanineafter hydrolysis. So the carnosine-like peak might not be a single peptide.Before and after hydrolysis, 1- and 3-methylhistidine were not found.The concentrations of ethanolamine and glutathione were extremely highin liver. Several unknown peptides which disappeared after hydrolysis wereseen as follows: several peptides which immigrate before aspartate in FractionsI, II, and III; one peptide before glysine in Fraction III; one peptide afterglutamate in Fraction II; one peptide after serine in Fraction 1.a·Aminobutyric acid (8.5/1. moles/ 100 g) was seen in Fraction I and didnot increase after hydrolysis.Glutamate and glycine were prodominant amino acids in Fraction IV, andsmall amounts of aspartate, threonine, serine, proline, and alanine were alsodetected.3Azumi: Studies on nitrogen pool of animal tissues. 3. Ox liver and bileProduced by The Berkeley Electronic Press, 1967324II. Ox Bladder BileT. AZUMIThe concentration of ampholytes in ox bladder bile was very low on thewhole.As in the case of liver, free arginine content was negligible. But unlikeliver, ornithine content was not high. After hydrolysis of Fraction I, incrementsof arginine and ornithine were very low, but that of histidine was somewhathigher and of lysine was moderate. An unknown peptide which overlapped onthe descending curve of lysine might be composed of lysine and histidine.Carnosine was found in the order of 2.0 fl. moles/lOa ml. B-Alanine COn-centration was 0.48 fl. mole before hydrolysis, and increased to 2.45 p. molesafter hydrolysis. t-Aminobutyric acid was not detected before and after hydro-lysis. I·Methylhistidine was found in trace amount after hydrolysis.Glutathione was not found in bladder bile, but it has been found occasionallyin the fistula bile of some animals in this laboratory.Many acidic peptides which appeared ,quite faster than aspartic acid, threepeptides which appeared before and after glutamic acid, and one peptide whichappeared after alanine, were seen in Fraction II. After hydrolysis of FractionII, glycine, glutamin, cystine, threonine, and serine increased. The peptidewhich appeared after alanine might be cystinyl glycine.Among amino acids in the bladder bile, glycine content was overwhelminglyhigh and followed by glutamic acid.In Fraction IV, which was the hydrolysates of N-covered ampholytes, gly-cine content (41.7 f.L moles/lOa ml) was also predominant and the other aminoacids were negligible. Taurine content was high only in Fraction I and was notfound in Fraction IV. A greater part of taurine might be excreted in urine.III. Ox KidneyAs reported already of guinea pig kidneyS, DCEC was found also in oxkidney in the order of O.l1p. mole/lOa g. CMC was also identified when thehydrolysate of Fraction II was fractionated on a long Diaion SK-l column asdescribed in the case of liver. Its amount was 0.19 f.L mole/laO g.Glutathione content of kidney was very low. This may be due to a strongglutathionase activity of kidney. A fairly large amount of carnosine (about 45/1moles/lOa g) was detected. The amount of B-alanine was 8.64 fl. moles/IOO gmbefore hydrolysis and 85.20 fl. moles/ 100 g after hydrolysis. i'-Aminobutyric acidincreased about 10 p. moies/IOO g after hydrolysis as shown in Table 1. Anserine-like small peak was seen and I-methylhistidine was found only after hydrolysisin the order of 13.06 p. moles/lOa g. A very small amount of 3-methylhistidinewas detected in the same order before and after hydrolysis.4Acta Medica Okayama, Vol. 21 [1967], Iss. 6, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol21/iss6/5Nitrogen Pool of Animal Tissues 325A small amount of hydroxylysine-like peak was found before hydrolysisand somewhat increased after hydrolysis.Some unknown peptides were seen as follows: in Fraction I, after urea;in Fraction II, three peptides before aspartate and two peptides before and afterglycine; in Fracsion III, several peptides before aspartate and two peptidesbefore and after glutamate. One peptide in Fraction II which appeared afterglycine in a fairly large amount might be cysteinylglycine. After hydrolysis ofFraction II, the increments of glycine and half cystine were almost in the sameorder (about 20 p. moles/lOa g).After hydrolysis of Fraction I, an unknown peak which had the sameheight as serine was seen just after serine.In Fraction IV, glycine and glutamate were usually predominant in theother tissues, but aspartate, serine, threonine, and alanine were found in a fairlylarge amount in ox kidney.IV. Ox LungOCEC and CMC were not detected in this tissue. The concentrations offree lysine, arginine, and glycine in lung were somewhat higher than in theother tissues. After hydrolysis of Fraction I, lysine and ethanolamine wereincreased remarkably, and an unknown peptide which immigrated before orni-thine disappeared. The amounts of a-alanine and r·aminobutyric acid were intrace and not increased after hydrolysis, but hydroxylysine-like small peakappeared after hydrolysis. Carnosine was not seen and 1- and 3-methylhistidinewere not detected before and after hydrolysis.Glutathione content was moderate and some other unknown peptides wereseen as follows: in Fraction II, four peptides before asparate and two peptidesbefore and after glycine; in Fraction III, several peptides before aspartate.A small amount of a-aminobutyric acid-like peak was seen in Fraction Ibefore hydrolysis, and in Fraction II after hydrolysis.In Fraction IV, aspartate, serine, threonine, glutamate, proline, glycine,and alanine were the major compounds.In general, lung may be said to be a featureless tissue in the nitrogen pool.DISCUSSIONDCEC and CMC have been found in animal urine\ and they were foundalso in some ox tissues. Especially DCEC was distributed widely in the animaltissues. Studies on the physiological significance and the biosynthetic pathwaysof DCEC and CMC will be carried in future.Although the following facts were not described in the results, a fairly large5Azumi: Studies on nitrogen pool of animal tissues. 3. Ox liver and bileProduced by The Berkeley Electronic Press, 1967326 T. AZUMIamount of an unknown amino acid which overlapped on the ascending curve ofaspartate was found in the hydrolysates of liver Fraction II and of bile Fraction1. Authentic erythro·fi-hydroxyglutamic acid just overlapped on aspartate butnot on the unknown. Authentic threo-fi-hydroxyglutamic acid emerged fasterthan the unknown.Some distinctive features of each tissue and fluid may be summarized asfollows. Free lysine and arginine contents were low, and the contents of orni-thine, ethanolamine, and glutathione were extremely high in liver. Some ofthese facts are suggestive of active urea cycle. The amino acid content of bilewas very low, and glycine was a predominant constituent. The nitrogen poolof kidney contained all the amino acids moderately, but glutathione content waslow. The latter may be due to a strong glutathionase activity of kidney. Thereare few reports on the nitrogen pool of lung. It was high in free arginine andlysine, and low in ornithine. In general, when histidine content was high, thecontents of lysine and arginine were low and vice versa.SUMMARYThe compositions of nitrogen pools of ox liver, bladder bile, kidney andlung were analyzed with an especial bearing on their minor components, andsome distinctive features of these tissues were described.DCEC and CMC were found in ox liver and kidney.Liver was low in free arginine and lysine, but high in ornithine, ethano-lamine, and glutathione.Glycine was only a predominant amino acid in ox bile.All amino acids were contained moderately in kidney, but glutathione con-tent was low.The concentrations of arginine and lysine were relatively high in lung.REFERENCES1. AZUMI, T.: Acta Med. Okayama 21, 19682. MIZUHARA, S.: "Protein-Nucleic Acid-Enzyme" 12, 308, 19673. KUWAKI, T., and MrzuHARA, S.: Biochim. Biophys. Acta 115, 491, 19664. UBUKA, T., KODAMA, H.. and MrzuHARA, S.: Biochim. Biophys. Acta 141, 266, 19675. CALAM, D. H.. and WALEY, S. G.: Biochem. ]. 86, 226, 19636Acta Medica Okayama, Vol. 21 [1967], Iss. 6, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol21/iss6/5

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