Some bile acids (dehydrocholic, cholic, chenodeoxycholic, ursodeoxycholic, and deoxycholic acids), and some hypocholesterolemic agents (22, 25 diazacholestanol, 20,25-diazacholesterol, triparanol, and SKF 525-A) are the inducers of isovalthinuria in guinea pig. Administration of methionine appears to increase
the pool of sulfur compound which participates in the formation of isovalthine. Cholesterol appears to have no enhancing effect on the induction activity of isovalthinuria inducers. The mechanism of isovalthine formation and the role of sulfur amino acids in lowering blood cholesterol are discussed.</p

Ubuka, Toshihiko

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Acta Medica OkayamaVolume 17, Issue 6 1963 Article 21963Experimental isovalthinuria. III. Induction bybile acids, and hypocholesterolemic agentsToshihiko Ubuka∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Experimental isovalthinuria. III. Induction bybile acids, and hypocholesterolemic agents∗Toshihiko UbukaAbstractSome bile acids (dehydrocholic, cholic, chenodeoxycholic, ursodeoxycholic, and deoxycholicacids), and some hypocholesterolemic agents (22, 25 diazacholestanol, 20,25-diazacholesterol,triparanol, and SKF 525-A) are the inducers of isovalthinuria in guinea pig. Administration ofmethionine appears to increase the pool of sulfur compound which participates in the formationof isovalthine. Cholesterol appears to have no enhancing effect on the induction activity of iso-valthinuria inducers. The mechanism of isovalthine formation and the role of sulfur amino acidsin lowering blood cholesterol are discussed.∗PMID: 14168971 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 17, 273-278 (1963)EXPERIMENTAL ISOVALTHINURIA*Ill. INDUCTION BY BILE ACIDS, ANDHYPOCHOLESTEROLEMIC AGENTSToshihiko UBUKADepartment of Biochemistry Okayama University Medical SchoolOkayamaReceived for publication, November 29, 1963For the elucidation of a question why some of the hypercholesterolemicpatients excrete isovalthine1•2 in their urine, a series of research has been con-ducted on the problem how the isovalthinuria can be induced in some experi-mental animals. FUKuToME3 has reported that oral or parenteral administrationof isovaleric acid induces isovalthinuria in some animals which never excrete iso-valthine in their normal urine. Isovaleric acid-I-CH administered, however, hasnot significantly incorporated into urinary isovalthine in dog".Recently some other reproducible methods for the induction of isovalthinuriahave been found in guinea pig by using bile acids or hypocholesterolemic agents.1. Experimental AnimalGuinea pigs are used as the experimental animal throughout this workbecause of the following reasons. 1. They never excrete isovalthine in theirnormal urine. 2. They excrete proper volume of urine and the size of their bodyis suitable for the future isotopic experiments. 3. They contain much glutathionein the body as compared to other animals5, and S-(isopropylcarboxymethyl)-glu-tathione synthesized in liver is assumed to be a direct precursor of urinary iso-valthine6•Male guinea pigs of approximately 3 months of age weighing 300-400gm were fed on RC·5 solid food (Oriental Yeast INC., Tokyo), and green vege-tables. L-Ascorbic acid (2.5 mg per animal) was supplied every other day by mix-ing in drinking water. Temperature of the animal room was conditioned ataround 25°C all the year round. Two guinea pigs were placed in a cage and theurine was collected once every week in a bottle containing toluene and hydro-chloric acid. Average volume of urine was around 500 ml per two guinea pigsper week.Bile acids used as inducers were dehydrocholic, cholic, chenodeoxycholic,ursodeoxycholic, and deoxycholic acids. Hypocholesterolemic agents used were* Supported by research grant., from the U. S. National Institutes of Health (HE-07419) andthe Ministry of Education of Japan.2731Ubuka: Experimental isovalthinuria. III. Induction by bile acids, andProduced by The Berkeley Electronic Press, 1963274 T. UBUKA22, 25-c1iazacholestanol (SC-11952), 20,25-diazacholesterol (SC-12937), tripara-nol (MER-29, 1-(p-(B-diethylaminoethoxy)-phenyl}1-(p-tolyl)-2-(p-chlorophenyl)-ethanol), and B-diethylaminoethyl-diphenylpropylacetate hydrochloride (SKF 525-A). Five mg of each bile acid and two mg of each azasterol were orallyadmi-nistered with powdered RC-5 solid food per animal per day but not per Kg bodyweight, because body weight of each guinea pig gained about 100 gm every fourweeks during experiments. Triparanol and SKF 525-A, however, were admi-nistered in 4.2 mg and 5 mg per Kg body weight per day respectively by weighingthe animal every two days.The influences of cholesterol (100 mg/animal/day) and of methionine (5mg/animal/day) on isovalthinuria induced by several inducers were also testedIn some cases.2. Preparation of Urinary Acidic Amino Acid Fraction for the Determina-tion of Isovalthine on Amino Acid Analyzer.For the determination of urinay isovalthine on an amino acid analyzer, iso-valthine in the urine must be concentrated beforehand.Two hundred ml of urine is made weakly acidic with acetic acid or ammoniaand filtered. The filtrate is transferred on a column containing 200 ml of Amber-lite AG-45 (acetate-form). The column is washed with one liter of 0.15 N aceticacid and eluted 2 liters of 2 N acetic acid. The 2 N acetic acid eluate is directlytransferred on a column containing 100 ml of Diaion SK-1 (H-form, strong cationexchanger, Mitsubishi Kasei Co. Ltd., Tokyo). The column is washed withdeionized water and then eluted with 500 ml of 2 N ammonia. The -ammoniasolution is evaporated to dryness in vacuum under 40°C. The residue is dissolvedin 8 ml of water and filtered. After isovalthine being identified in the filtrateaccording to the author's method7, two ml of the filtrate corresponding to 50 mlof original urine is analyzed on amino acid analyzer (Beckman Model 120 B).When the sample for the analyzer contains too much dirty substances, the driedresidue above prepared is treated as follows. The residue is dissolved in about10 ml of 0.15 N acetic acid and filtered. The filtrate is passed through a columncontaining 10 ml of Amberlite CG-50 (H-form), and the column is washed with50 ml of 0.15 N acetic acid. The effluent and washings are combined and driedin vacuum. This procedure is useful for decolorization of the samples and therecovery of urinary isovalthine is not reduced.Amino acid analyzer is operated with 0.2 N sodium citrate buffer (pH 3.24)at 30°C on 150 cm column throughout this work. The urinary isovalthine (L-isovalthire*) yields a single peak between serine and proline at around 196 e££lu-*Details on the configuration and synthesis of urinary isovalthine will be shortly reportedelsewhere by S. Ohmori of this Laboratory.2Acta Medica Okayama, Vol. 17 [1963], Iss. 6, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol17/iss6/2Experimental lsovalthinuria 275ent ml and it:; HW-constant is 20.5. The preparation procedure of urinary acidicamino acid fraction described above never changes the configuration of urinaryisovalthine and the recovery of urinary isovalthine is around 75 % which is usedfor the calculation in this experiment.3. Induction of Isovalthinuria by Bile Acids.The results obtained by feeding with several bile acids are summarized inTable 1. Most of the guinea pigs received dehydrocholic acid begin to excreteisovalthine in 5th-7th day after the start of administration, and the maximumexcretion is observed on the second week and decreased thereafter (Table 1. a, c,d). A group of guinea pigs receiving dehydrocholic acid (Table 1. b), however,showed the maximum excretion on the fourth week as in the cases of the otherbile acids (Table 1. e, g, h, i). Since the amount of isovalthine excretion variesconsiderably from one animal to another, it is not certain at present whethercholesterol enhances the induction effect of bile acids or not (Table 1. d, f). Ad-ministration of cholesterol alone (100 mg/animal/day) has never induced isoval-thinuria up to the end of fourth week. When the excretion of isovalthine becameto zero after passing the maximum peak of excretion, the administration of me-thionine only on the sixth week induced again isovalthinuria (Table 1. c, e, h).After methionine administration on the sixth week, no isovalthinuria was ob-served by continued feeding with dehydrocholic acid up to the end of eleventhweek (Table 1. c). But methionine administration on the twelfth week inducedagain isovalthinuria (Table 1. c). Isovalthine excretion on the fifth week follow-ing methionine administration is also considered to be the methionine effect(Table 1. b, g), because most animals having the maximum excretion on thefourth week do not excrete isovalthine on the fifth week unless methionine isgiven (Table 1. e, h, i).Table 1 Experimental Isovalthinuria Induced By Bile Acids.Week I /l moles of isovalthine excreted by two guineapigs per week./I~I~I~I~I~I~IN~I~Ii~-Dehydrocholic acid I 1.56 3.24 2.50 2.111 1 .48 - - -IDehydrocholic acid 0.00 0.00 0.99 12.041 4.01* - - -Dehydrocholic acid 15.35 66.52 10.57 2.80 0.00 1.65* 0.00 4.40*Dehydrocholic acid + Cholesterol 2.62 6.60 2.58 0.93 - - - -Cholic acid 0.00 0.00 0.00 2.80 0.00 28.15* - -Cholic acid + Cholesterol 0.00 0.00 2.45 0.00 - - - -Chenodeoxycholic acidI0.00 0.00 0.99 11.25 1.80* - - -Ursodeoxycholic acid 0.00 0.00 3.44 14.40 0.00 37.44* - -Deoxycholic acidIO. 00 0.00 0.00 4.05 0.00 0.00* - -Ib.e.1.d.a.f.g.h.c.* Methionine (5mg/animal/day) is administered for seVen days on the sited week only.3Ubuka: Experimental isovalthinuria. III. Induction by bile acids, andProduced by The Berkeley Electronic Press, 1963276 T. UBUKASince continued feeding with methionine alone (5 mg/animal/day) has notinduced isovalthinuria at least up to the end of fourth week after the start ofadministration, the above phenomena may be considered to indicate that thefeeding with bile acids is accompanied by the reduction of sulfur compounds inthe animal body. No methionine effect observable in the case of deoxycholic acidcannot be explained at present (Table 1. n.4. Induction of Isovalthinuria by Hypocholesterolemic AgentsThe results obtained by feeding with hypocholesterolemic agents are sum-marized in Table 2. Triparanol and' 22, 25·diazacholestanol can induce isoval-thinuria having the maximum peak of excretion on the third week (Table 2. a,b, and d). Cholesterol appears to have no influence on the induction effect ofazasterol (Table 2. b). Although induction effect of 20, 25-diazacholesterol seemsto be quite weak, a large amount of isovalthine has been excreted following theadministration of methionine on the sixth week (Table 2. c). This phenomenonmay also indicate that the administration of azasterol is accompanied by the de-pression of sulfur compounds in the animal body. SKF 525-A exhibits very weakinduction activity barely on the sixth week, and methionine administration onthe senventh week has increased isovalthine excretion (Table 2, e).Table 2 Experimental Isovalthinu~ia Induced By Hypocholesterolemic Agents.-------.. Week /.I. moles of total isovalthine excreted by two guinea pigs per week.....ducers I! I Ii 6th I 7th- 1st 2nd ! 3rd 4th 5th I--I22, 25-DiazacholestanolI0.00I2.05 2.35 I 0.00\--I-I22,25-Diazacholestanol 0.00 1. 08 1.28 I 0.00 ! -20·:.1-+ CholesterolI20, 25-Diazacholesterol 0.00 0.00 0.00 0.60 : 0.00 -Triparanol 0.00 1.71 20.40 I 0.00 : - -I 5.~* 1SKF 525-A I I 0.00 0.75 i0.00 0.00 0.00 I 0.00Inedcba* Methionine (5 mg/animal/day) is administered for seven days on the sited week only.DISCUSSIONIt has heen reported that bile acids inhibit the biosynthesis of cholesterol bya double feedback mechanisms. Chenodeoxycholic and 3-hydroxy-7-oxocholanicacids are known to be the main constituents of the bile of guinea pig, andcholic acids is said to be contained in the adult bile onll. But there appears tobe no difference in the induction activity between constituents and non-consti-tuents of the bile. The amount of urinary isovalthine following the administra-tion of these bile acids may rather depend on the individual constitution of eachanimal.4Acta Medica Okayama, Vol. 17 [1963], Iss. 6, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol17/iss6/2Experimental lsovalthinuria 277Since hypocholesterolemic agents tested here are said to inhibit some enzy-matic steps of cholesterol biosynthesis1o.ll,l'2,13 it may be considered that treatmentof animals with these bile acids or those hypocholesterolemic agents will lead tothe accumulation of cholesterol precursors such as isovaleric acid etc. and thisaccumulation of precursors will then lead to the formation of isovalthine by con-jugating with sulfur compound. This consideration, however, is not certain atpresent, because some preparations of glycocorticoids and ACTH also induceisovalthinuria.*The latter findings might suggest also a correlation of isovalthinewith stress. At any rate, further experiments will be needed for the elucidationof the exact meaning of isovalthine excretion.The sulfur compound which conjugates directly with isovaleric acid or re-lated compound is now considered to be glutathione from the experiment ofKUWAKI et a1.6• Methionine administered will fortify the concentration of gluta-thione in animal body or may also activate some SH enzymes which mightparticipate in the synthetic reaction of isovalthine precursor (glutathione-iso-valeric acid conjugate). Although it is only a single experiment in this labora-tory, the experiment shows that an atherosclerotic patient having no isoval-thinuria has excreted isovalthine 'Nith a concomitant decrease of blood cholesterolafter receiving methionine. Thus isovalthine formation might have a correlationwith some reports14,lO which show the blood cholesterol lowering effect of somesulfur amino acids.SUMMARYSome bile acids (dehydrocholic, cholic, chenodeoxycholic, ursodeoxycholic,and deoxycholic acids), and some hypocholesterolemic agents (22, 25-diaza-cholestanol, 20,25-diazacholesterol, triparanol, and SKF 525-A) are the inducersof isovalthinuria in guinea pig. Administration of methionine appears to increasethe pool of sulfur compound which participates in the formation of isovalthine.Cholesterol appears to have no enhancing effect on the induction activity ofisovalthinuria inducers. The mechanism of isovalthine formation and the role ofsulfur amino acids in lowering blood cholesterol are discussed.ACKNOWLEDGMENTThe author wants to thank Prof. Dr. S. Mizuhara for his interest and support duringthis work.REFERENCES1. MIZUHARA, S., and OHMORI, S.: Arch. Biochem. BioPhys., 92, 53 (l961).2. OHMORI, S., and MIZUHARA, S.: Arch. Biochem. Biophys., 96, 179 (l962).3. FUKuToME, K.: Acta Med. Okayama, 16, 241 (1962).* Unpublished data of this L'Iboratory.5Ubuka: Experimental isovalthinuria. III. Induction by bile acids, andProduced by The Berkeley Electronic Press, 1963278 T. UBUKA4. FUKUTOME, K.: Acta Med. Okayama, 16, 247 (1962).5. West, E. S., and HIGHET, D. M.: Proc. Soc. Exptl. BioI. Med., 68, 60 (1948).6. KUWAKI, T., OHMORI, S., and MIZUHARA, S.: Biochem. Biophys. Acta, (1963) in press.7. UBUKA, T.: ]. Biochem., 52. 440 (1962).8. BEHER, W. T., Baker, G. D., and ANTHONY, W. L.: Proc. Soc. Exptl. Bioi. Med.,109, 863 (1962).9. PERIC-GOLIA, L., and JONES, R. S.: Proc. Soc. Exptl. Bioi. Med., 105, 337 (1960).10. RANNEY, R. E., and Counsell, R. E.: Proc. Soc. Exptl. Bioi. Med., 109, 820 (1962).11. KLEIN, P. D., RANNEY, R. E., CoUNSELL, R. E., and SZCEPANIK, P. A.: Fed. Proc., 22,591 (1963).12. DICK, E. C., GREENBERG, S. M., HERNDON, J. F.. JONES, M., and VAN Loon, E. J.:Proc. Soc. Exptl. Bioi. Med., 104, 523 (1960).13. AVIGAN, ]., STEINBERG, D., VROMAN, H. E., THoMPsoN, M. J., and MOSETTIG, E.: ].Bioi. Chem., 235, 3123 (1960); STEINBERG, D., and AVIGAN, J.: ]. Bioi. Chem., 235,3127 (1960).14. PORTMAN, O. W., and MANN, G. V.: ]. Bioi. Chem., 213, 733 (1955).15. SEIDEL, .T. C., and HARPER, A. E.: Proc. Soc. Exptl. Bioi. Med., 111, 579 (1962).6Acta Medica Okayama, Vol. 17 [1963], Iss. 6, Art. 2http://escholarship.lib.okayama-u.ac.jp/amo/vol17/iss6/2

Experimental isovalthinuria. III. Induction by bile acids, and hypocholesterolemic agents

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