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Experimental isovalthinuria — Induction by hormones
325
CLINIC.4 CHIMICA ACTA
EXPERIMENTAL
ISOVALTHINURIA*
-
INDUCTION
BY HORMONES
I<. HORIUCHI Department
(Received
ofBiochemistry,
Okayallza
University
Medical
School, Okayama
(Japan)
October gth, 1964)
SUMMARY
Remarkable isovalthinuria can be induced in guinea pigs by the administration of prednisolone, dexamethasone, ACTH, or epinephrine, but not by that of deoxycorticosterone, testosterone, estradiol, progesterone, insulin, or choline. dl-qMethyltestosterone seems to have a weak inducing effect. The inducing activity of dexamethasone or ACTH is counteracted by insulin.
INTRODUCTION
Isovalthinel has been found in the urine of normal cats and hypercholesterolemic patients such as those having myxedema, diabetes, and atherosclerosis. The chemical structure2 and the configuration3 of urinary isovalthine have already been established. CH, HOOC-CH-CHz-S-CH--CA
I NH,
I
COOH
‘CH,
As noted below, there are many indications which suggeSt an intimate relation between isovalthine and cholesterol metabolism. Besides isovalthine, cats also excrete felinine4 in their normal urine and cats are known to be the most unsuitable animal for the induction of experimental atherosclerosis. Isovaleric acid, a constituent of the isovalthine molecule, is a precursor for cholesterol biosynthesis5, and the isoamyl alcohol residue of felinine is derived from mevalonic acids. Administration of isovaleric acid ‘I, some bile acids 8, and some hypocholesterolemic agents8 induces isovalthinuria in some animals which never normally excrete isovalthine in their normal urine. In these studies on experimental isovalthinuria, * This work was supported by the research grants from the U.S. National (HE-07419) and the Ministry of Education of Japan. Clin. Chim.
Institutes
Acta,
12
(1965)
of Health
325-329
K. HORIUCHI
326
the administration of methionine or cysteine enhances isovalthine excretion*, and 3% of these sulfur amino acids is incorporated into urinary isovalthineg. In this connection, it is worthy of notice that some sulfur-containing amino acids are reported to be capable of lowering the blood cholesterol levell’J> Il. Isovalthine is now considered to be synthesized at first as a glutathione-isovaleric acid conjugate (GSIV) 12, which is converted into isovalthine in kidney 13. In spite of our knowledge of these points, there still remain many obscurities on the mechanism of isovalthinuria induction. Since hypercholesterolemic patients mentioned above are in a hormonal imbalance, some hormones are assumed to be responsible for the induction of isovalthinuria. This assumption has now been tested by using several hormones and the results obtained here appear to indicate that the glucocorticoids play a central role for the induction of isovalthinuria. MATERIALS
AND
METHODS
.4nimals Male guinea pigs weighing 350-450 g were used as the experimental animals throughout this work. Two guinea pigs were placed in each cage and the urine was collected once a week in a bottle containing toluene and acetic acid. The basal diet was RC-5 solid food (Oriental Yeast Inc., Tokyo) and green vegetables. Ascorbic acid (1.0 mg/animal/day) was supplied in the drinking water. Room temperature was maintained at 20-23~ in winter and around 25” in summer. I.
Hormones Hormones tested as inducers were prednisolone phosphate (“Codelsol”, Banyu Pharmaceutical Co. Ltd., Tokyo), dexamethasone (“Corsone”, Takeda Chem. Ind. Ltd., Osaka), ACTH (“Acthar”, Armour Pharmaceutical Co., U.S.A.), epinephrine (“Adrenalin”, Sankyo Co. Ltd., Tokyo), deoxycorticosterone acetate (Syncorta”, Takeda), ,4 1-17-methyltestosterone (“Abirol”, Takeda), testosterone propionate (“E narmon”, Teikoku Hormone Mfg. Co, Ltd., Tokyo), estradiol benzoate “Ovahormone Benzoate”, Teikoku Hormone), progesterone (“Oophormin Luteum”, Teikoku Hormone), insulin (“Iszilin”, Shimizu Pharmaceutical Co. Ltd., Shimizu), and choline. Glucocorticoids are administered per OS and the other hormones are injected subcutaneously. The amount of hormones used is listed in Table I. They are administered per animal per day but not per kg body weight, because body weight of each guinea pig gains about IOO g every four weeks during the experiments. 2.
3. Determination of urinary isovalthine Two hundred ml of collected urine were made weakly acidic with acetic acid and filtered. The filtrate was transferred to a column containing 200 ml of Amberlite IR-45 (acetate form). The column was washed with I 1 of 0.15 N acetic acid and eluted with 2 1 of 2 N acetic acid. The 2 N acetic eluate was directly transferred to a column containing 50 ml of Diaion SK-I (H-form, strong cation exchanger, Mitsubishi Kasei Co. Ltd., Tokyo). The column was washed with deionized water and then eluted with z X ammonia. The ammonia eluate was dried in vacua below 40’. The urinary acidic amino acid fraction thus obtained was dissolved in 4 ml of water and filtered. After isovalthine had been identified in the filtrate according to the method of Ubuka14, Clilz. Chim.
Acta,
IZ (1965) 3~5-3~9
ISOV_.lLTHINURIA
I
INDUCTION
BY HORMONES
ml of the filtrate corresponding
valthine
on an automatic
327
to 50 ml of the original urine was determined
amino acid analyzer
(Beckman
Model
for iso-
B) which was
120
operated with 0.2 AT sodium citrate buffer (pH 3.24) at 30’ on 150 cm column. Usually urinary L-isovalthine yields a single peak between glutamine and proline at around rg7 effluent ml and its HW-constant is 20.5 (cf.“). For some unknown reason, however, urinary isovalthine yields sometimes two adjacent peaks with the effluent ml at 184 and 197. The 184ml peak is L-alloisovalthine and its HW-constant is 19.3 (cf3). RESULTS
The results are summarized in Table I. Among the hormones tested here, glucocorticoids and ACTH seem to have the strongest activity for the induction of isovalthinuria. Dexamethasone was much more effective than prednisolone. In contrast to the glucocorticoids, ACTH action continues some time after its administration has been stopped, so the inducing effect of ACTH may be considered to be mediated through glucocorticoids. Insulin seems to antagonize, by an unknown mechanism, the inductive activity of glucocorticoids and of ACTH. Epinephrine has a weak but definite inductive activity. According to the stress theory, the epinephrine effect might also be mediated through ACTH and then glucocorticoids. Subcutaneous injection of epinephrine could not be continued so long, because it caused stiffness of the animals’ skin. Deoxycorticosterone, progesterone, and sex hormones, male or female, had little or no inducing activity, but d 1-r7-methyltestosterone, which is regarded as a protein anabolic hormone, showed a weak activity. In a previous investigations of the inductive action of bile acids or hypocholesterolemic agents, a maximum peak of isovalthine excretion was observed about the fourth week after the start of its administration, but no such distinct peak was observed in the cases of glucocorticoids or ACTH. After passing the maximum peak of isovalthine excretion in the cases of bile acids or hypocholesterolemic agents, the administration of methionine (5 mg/animal/ day) for just one week caused again a remarkable isovalthinuria*. No such methionine effect was observed in the cases of hormones. The administration of methionine alone (5 mg/animal/day) does not induce isovalthinuria up to the end of the fourth week, but after that it induces isovalthinuria as follows: 5th6th week, trace; 7th, 1.00 pmole; Bth, 17.25 pmoles; 9th (no methionine administration), 10.25 pmoles/two guinea pigs/week. In another experiment with methionine alone: rst-4th week, 0.00; 5th, 1.58 ,umoles; 6th, 5.14 pmoles; 7th, 1.45 pmoles/two guinea pigs/week. For the elucidation of the question why prolonged feeding of methionine induces isovalthinuria, choline was tested for its inducing effect, but it showed no inducing activity, as shown by Table I.
Although the mechanism for the induction of isovalthinuria by glucocorticoids is not clear at present, the following facts will be of value for the advancement of isovalthine studies. Clin. Chim. Acta,
12
(1965)3y,-3zg
‘% 0
% “;
c”
al
z
$ b c P _
c, L
I
unit unit unit unit unit mg
0.00
0.00
0.00
0.00
0.0”
0.00 2.20 + 0.00 0.00 0.00 0.00 + 0.00 0.00 0.00 0.00 0.00
0 00 0.00 0.00 0.00 0.00 + 0.00 0.00 0.00 0.00 0.00 0.00 0.00
2.23
6.32 3.28 29.15 0.00
+
‘+@t 0.00 I.&+* 0.00 0.00 + 0.00 o.oo* 0.00 0.00 0.00
+ 7.89 13.98 0.00 I .08 0.00 1 0.00 0.00 0.00 0.00 0.00
0.00 16.22 5.55 0.00 0.00 0.00 0.00 + 0.00 0.00 0.00 0.00 0.00
2.83
+ 12.87
7.4354 35.o9t 0.00
-
2.50 28.01 37.17 0.00
6.101
6th
pips per week
5.12 9.75 23.27 12.76 35.30 0.00
5th
by two pinea
4th
Positive on paper electropherogram but negative on amino acid analyzer. 1Mcthioninc (5 mg/animal/dap) is administered for 7 da>;s on the cited week only. Stop the administration of hormones at the week end &cd.
mg mg mg unit mg
excreted
3rd
3.61 + ‘7.15 6.56
0.00
+
and
+ * t
2.5 2.5 2.5 0.5 10.0
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0 00
0.00
0.00
0.00
0.00
0.00
0.00
rst
~~Moles of isovulthim
Testosterone Estradiol Progesterone Insulin Choline
0.1 mg 0.5 mg 0.5 mg
0.5 I .o I.0 1.0 0.5 0.1
0.1 mg 1.0 mg 0.1 2.5 mg mg 0.2 mg 0.2 mg 0.5 unit
Dose per animal per day
g. IO. 11. * 2. 13.
8. A’-17-Methyltestosterone
7. Deoxycorticosterone
5. ACTH + Insulin 6. Epinephrine
4. ACTH
3. Dcxamethasonc + insulin
2. Dexamethasone
I. Prednisolone
Hormones
~
EXPERIMENTALISO”ALTHIN”RIA INDUCED BY HORMONES
TABLE
-
-
0.00 0.00
o.oo*
o.oo* o.oo*
1::::’ -
+
-
3.50
20.45
+* -
7th
-
-
-
0.00
‘7.72 7.35
5.33t
0.00
25.5ot
-
8th
8.92 -
-
2.83
-
9th
-
ISOV.%LTHINURIA
INDUCTION
329
BY HORMONES
I.Glucocorticoids are known to be capable of elevating blood cholesterol level in some animals. 2. Glucocorticoids are known to antagonize insulin and diabetic patients excrete isovalthine. 3. AWholestenone is known to have a strong inhibitory effect on cholesterol biosynthesis’5 and to cause adrenal hypertrophy16. When guinea pigs are fed A4-cholestenone (IOO mg/animal/day), isovalthinuria has been induced as follows: rst-3rd week, 0.00; 4th, trace; 5th, 2.40 pmoles; 6th (methionine administered 5 mg/animal/ day), 2.46 ,umoles/two guinea pigs/week. Unexpectedly, however, the administration of cholesterol alone (200 mg/animal/day} never induces isovalthinuria up to the end of fourth month. 4. Bile acids are known to inhibit cholesterol biosynthesis by a double feedback mechanism I’, and in our experiences, adrenal hypertrophy has sometimes been observed in guinea pigs after feeding bile acids for several weeks. The inducing activity of dehydrocholic acid was also antagonized by insulin (unpublished data). 5. Since glucocorticoids are known to accelerate the deamination of some amino acids, isovaleric acid formation from leucine might also be increased. Administration of isovaleric acid can induce iso~~althin~Iria in some animals without exception?. At any rate, further studies are needed for the elucidation of the mechanism of isovalthine production in hypercholesterolemic patients. ACKNOWLEDGEMENT
The continued interest of Prof. Dr. S. Mizuhara in this study is gratefully acknowledged. REFERENCES AND S. OHMORI, Arch. BiocAem. Biophvs., gz (1961) 53. AND S. MTZUHARA, A&. B~~che~~.B~o~k~s., 96 (1~62) 179. Arch. Biochem. Bioph_vs., 104 (1964) 509. R. G. WESTALL, B&hem. J.. 55 (1955) 244. I. Z.%RIN AND K. BLOCK, ,J, Biol. Chem., rgz (1951) 207 P. V. AVIZONIS AND J. C. WRISTON, Biochim. Biophys. Acta, 34 (1956) 279. I<. FUKUTOME, Acta Med. Ok~.yama, 16 (rgGz) 241. ;47. T. UBUKA, _4cta Med. Okuyama, 17 (1963) 273. T. TJBuK.~,K. HORIUCHI, T. SHIMOMUR~ AND S. MlzuH~R~,Acta~ed. Okayama, r8(rg64) 65. 0. W. PORTMAN AND G. V.MANN,J. Bid. Chem., 213 (1955) 733. J. C. SEIDEL AND A. E. HARPER, Proc. Sot. Exptl. Biol. Med., III (1962) 579. T. KUW~KI, S. OHMORI AND S. MIZUHARA, Biochim. Biophys. Acta, 78 (x963) 553. T. K~~AKI, J. Biochem., 57 (1965) 125. T. UBUKA, J. B&hem.. 52 (1962) 440. G. M. TOMKINS, H. SHEPPARD AND I. L. CHAIKOFF,J.B~OZ. Chem., 203 (1953) 781. D. STEINBERG AND D.S. FREDRICKSON, Al8n.N.Y. Acad.Sci.,64(rg56) 579. W. T. BEHER, G. D. BAKER AND W. L. ANTHONY, Proc. Sot. Exptl. Biol. Med., rog (rg6z) 863.
I S.MIZUHARA 2 S. OHMORI 3 S. OHMORI,
4 i
7 8 g
IO I T 12 13 14 15
16 77
Cl&z. Chim.
Acta,
12 (1965) 325-329
CLINIC.4 CHIMICA ACTA
EXPERIMENTAL
ISOVALTHINURIA*
-
INDUCTION
BY HORMONES
I<. HORIUCHI Department
(Received
ofBiochemistry,
Okayallza
University
Medical
School, Okayama
(Japan)
October gth, 1964)
SUMMARY
Remarkable isovalthinuria can be induced in guinea pigs by the administration of prednisolone, dexamethasone, ACTH, or epinephrine, but not by that of deoxycorticosterone, testosterone, estradiol, progesterone, insulin, or choline. dl-qMethyltestosterone seems to have a weak inducing effect. The inducing activity of dexamethasone or ACTH is counteracted by insulin.
INTRODUCTION
Isovalthinel has been found in the urine of normal cats and hypercholesterolemic patients such as those having myxedema, diabetes, and atherosclerosis. The chemical structure2 and the configuration3 of urinary isovalthine have already been established. CH, HOOC-CH-CHz-S-CH--CA
I NH,
I
COOH
‘CH,
As noted below, there are many indications which suggeSt an intimate relation between isovalthine and cholesterol metabolism. Besides isovalthine, cats also excrete felinine4 in their normal urine and cats are known to be the most unsuitable animal for the induction of experimental atherosclerosis. Isovaleric acid, a constituent of the isovalthine molecule, is a precursor for cholesterol biosynthesis5, and the isoamyl alcohol residue of felinine is derived from mevalonic acids. Administration of isovaleric acid ‘I, some bile acids 8, and some hypocholesterolemic agents8 induces isovalthinuria in some animals which never normally excrete isovalthine in their normal urine. In these studies on experimental isovalthinuria, * This work was supported by the research grants from the U.S. National (HE-07419) and the Ministry of Education of Japan. Clin. Chim.
Institutes
Acta,
12
(1965)
of Health
325-329
K. HORIUCHI
326
the administration of methionine or cysteine enhances isovalthine excretion*, and 3% of these sulfur amino acids is incorporated into urinary isovalthineg. In this connection, it is worthy of notice that some sulfur-containing amino acids are reported to be capable of lowering the blood cholesterol levell’J> Il. Isovalthine is now considered to be synthesized at first as a glutathione-isovaleric acid conjugate (GSIV) 12, which is converted into isovalthine in kidney 13. In spite of our knowledge of these points, there still remain many obscurities on the mechanism of isovalthinuria induction. Since hypercholesterolemic patients mentioned above are in a hormonal imbalance, some hormones are assumed to be responsible for the induction of isovalthinuria. This assumption has now been tested by using several hormones and the results obtained here appear to indicate that the glucocorticoids play a central role for the induction of isovalthinuria. MATERIALS
AND
METHODS
.4nimals Male guinea pigs weighing 350-450 g were used as the experimental animals throughout this work. Two guinea pigs were placed in each cage and the urine was collected once a week in a bottle containing toluene and acetic acid. The basal diet was RC-5 solid food (Oriental Yeast Inc., Tokyo) and green vegetables. Ascorbic acid (1.0 mg/animal/day) was supplied in the drinking water. Room temperature was maintained at 20-23~ in winter and around 25” in summer. I.
Hormones Hormones tested as inducers were prednisolone phosphate (“Codelsol”, Banyu Pharmaceutical Co. Ltd., Tokyo), dexamethasone (“Corsone”, Takeda Chem. Ind. Ltd., Osaka), ACTH (“Acthar”, Armour Pharmaceutical Co., U.S.A.), epinephrine (“Adrenalin”, Sankyo Co. Ltd., Tokyo), deoxycorticosterone acetate (Syncorta”, Takeda), ,4 1-17-methyltestosterone (“Abirol”, Takeda), testosterone propionate (“E narmon”, Teikoku Hormone Mfg. Co, Ltd., Tokyo), estradiol benzoate “Ovahormone Benzoate”, Teikoku Hormone), progesterone (“Oophormin Luteum”, Teikoku Hormone), insulin (“Iszilin”, Shimizu Pharmaceutical Co. Ltd., Shimizu), and choline. Glucocorticoids are administered per OS and the other hormones are injected subcutaneously. The amount of hormones used is listed in Table I. They are administered per animal per day but not per kg body weight, because body weight of each guinea pig gains about IOO g every four weeks during the experiments. 2.
3. Determination of urinary isovalthine Two hundred ml of collected urine were made weakly acidic with acetic acid and filtered. The filtrate was transferred to a column containing 200 ml of Amberlite IR-45 (acetate form). The column was washed with I 1 of 0.15 N acetic acid and eluted with 2 1 of 2 N acetic acid. The 2 N acetic eluate was directly transferred to a column containing 50 ml of Diaion SK-I (H-form, strong cation exchanger, Mitsubishi Kasei Co. Ltd., Tokyo). The column was washed with deionized water and then eluted with z X ammonia. The ammonia eluate was dried in vacua below 40’. The urinary acidic amino acid fraction thus obtained was dissolved in 4 ml of water and filtered. After isovalthine had been identified in the filtrate according to the method of Ubuka14, Clilz. Chim.
Acta,
IZ (1965) 3~5-3~9
ISOV_.lLTHINURIA
I
INDUCTION
BY HORMONES
ml of the filtrate corresponding
valthine
on an automatic
327
to 50 ml of the original urine was determined
amino acid analyzer
(Beckman
Model
for iso-
B) which was
120
operated with 0.2 AT sodium citrate buffer (pH 3.24) at 30’ on 150 cm column. Usually urinary L-isovalthine yields a single peak between glutamine and proline at around rg7 effluent ml and its HW-constant is 20.5 (cf.“). For some unknown reason, however, urinary isovalthine yields sometimes two adjacent peaks with the effluent ml at 184 and 197. The 184ml peak is L-alloisovalthine and its HW-constant is 19.3 (cf3). RESULTS
The results are summarized in Table I. Among the hormones tested here, glucocorticoids and ACTH seem to have the strongest activity for the induction of isovalthinuria. Dexamethasone was much more effective than prednisolone. In contrast to the glucocorticoids, ACTH action continues some time after its administration has been stopped, so the inducing effect of ACTH may be considered to be mediated through glucocorticoids. Insulin seems to antagonize, by an unknown mechanism, the inductive activity of glucocorticoids and of ACTH. Epinephrine has a weak but definite inductive activity. According to the stress theory, the epinephrine effect might also be mediated through ACTH and then glucocorticoids. Subcutaneous injection of epinephrine could not be continued so long, because it caused stiffness of the animals’ skin. Deoxycorticosterone, progesterone, and sex hormones, male or female, had little or no inducing activity, but d 1-r7-methyltestosterone, which is regarded as a protein anabolic hormone, showed a weak activity. In a previous investigations of the inductive action of bile acids or hypocholesterolemic agents, a maximum peak of isovalthine excretion was observed about the fourth week after the start of its administration, but no such distinct peak was observed in the cases of glucocorticoids or ACTH. After passing the maximum peak of isovalthine excretion in the cases of bile acids or hypocholesterolemic agents, the administration of methionine (5 mg/animal/ day) for just one week caused again a remarkable isovalthinuria*. No such methionine effect was observed in the cases of hormones. The administration of methionine alone (5 mg/animal/day) does not induce isovalthinuria up to the end of the fourth week, but after that it induces isovalthinuria as follows: 5th6th week, trace; 7th, 1.00 pmole; Bth, 17.25 pmoles; 9th (no methionine administration), 10.25 pmoles/two guinea pigs/week. In another experiment with methionine alone: rst-4th week, 0.00; 5th, 1.58 ,umoles; 6th, 5.14 pmoles; 7th, 1.45 pmoles/two guinea pigs/week. For the elucidation of the question why prolonged feeding of methionine induces isovalthinuria, choline was tested for its inducing effect, but it showed no inducing activity, as shown by Table I.
Although the mechanism for the induction of isovalthinuria by glucocorticoids is not clear at present, the following facts will be of value for the advancement of isovalthine studies. Clin. Chim. Acta,
12
(1965)3y,-3zg
‘% 0
% “;
c”
al
z
$ b c P _
c, L
I
unit unit unit unit unit mg
0.00
0.00
0.00
0.00
0.0”
0.00 2.20 + 0.00 0.00 0.00 0.00 + 0.00 0.00 0.00 0.00 0.00
0 00 0.00 0.00 0.00 0.00 + 0.00 0.00 0.00 0.00 0.00 0.00 0.00
2.23
6.32 3.28 29.15 0.00
+
‘+@t 0.00 I.&+* 0.00 0.00 + 0.00 o.oo* 0.00 0.00 0.00
+ 7.89 13.98 0.00 I .08 0.00 1 0.00 0.00 0.00 0.00 0.00
0.00 16.22 5.55 0.00 0.00 0.00 0.00 + 0.00 0.00 0.00 0.00 0.00
2.83
+ 12.87
7.4354 35.o9t 0.00
-
2.50 28.01 37.17 0.00
6.101
6th
pips per week
5.12 9.75 23.27 12.76 35.30 0.00
5th
by two pinea
4th
Positive on paper electropherogram but negative on amino acid analyzer. 1Mcthioninc (5 mg/animal/dap) is administered for 7 da>;s on the cited week only. Stop the administration of hormones at the week end &cd.
mg mg mg unit mg
excreted
3rd
3.61 + ‘7.15 6.56
0.00
+
and
+ * t
2.5 2.5 2.5 0.5 10.0
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0 00
0.00
0.00
0.00
0.00
0.00
0.00
rst
~~Moles of isovulthim
Testosterone Estradiol Progesterone Insulin Choline
0.1 mg 0.5 mg 0.5 mg
0.5 I .o I.0 1.0 0.5 0.1
0.1 mg 1.0 mg 0.1 2.5 mg mg 0.2 mg 0.2 mg 0.5 unit
Dose per animal per day
g. IO. 11. * 2. 13.
8. A’-17-Methyltestosterone
7. Deoxycorticosterone
5. ACTH + Insulin 6. Epinephrine
4. ACTH
3. Dcxamethasonc + insulin
2. Dexamethasone
I. Prednisolone
Hormones
~
EXPERIMENTALISO”ALTHIN”RIA INDUCED BY HORMONES
TABLE
-
-
0.00 0.00
o.oo*
o.oo* o.oo*
1::::’ -
+
-
3.50
20.45
+* -
7th
-
-
-
0.00
‘7.72 7.35
5.33t
0.00
25.5ot
-
8th
8.92 -
-
2.83
-
9th
-
ISOV.%LTHINURIA
INDUCTION
329
BY HORMONES
I.Glucocorticoids are known to be capable of elevating blood cholesterol level in some animals. 2. Glucocorticoids are known to antagonize insulin and diabetic patients excrete isovalthine. 3. AWholestenone is known to have a strong inhibitory effect on cholesterol biosynthesis’5 and to cause adrenal hypertrophy16. When guinea pigs are fed A4-cholestenone (IOO mg/animal/day), isovalthinuria has been induced as follows: rst-3rd week, 0.00; 4th, trace; 5th, 2.40 pmoles; 6th (methionine administered 5 mg/animal/ day), 2.46 ,umoles/two guinea pigs/week. Unexpectedly, however, the administration of cholesterol alone (200 mg/animal/day} never induces isovalthinuria up to the end of fourth month. 4. Bile acids are known to inhibit cholesterol biosynthesis by a double feedback mechanism I’, and in our experiences, adrenal hypertrophy has sometimes been observed in guinea pigs after feeding bile acids for several weeks. The inducing activity of dehydrocholic acid was also antagonized by insulin (unpublished data). 5. Since glucocorticoids are known to accelerate the deamination of some amino acids, isovaleric acid formation from leucine might also be increased. Administration of isovaleric acid can induce iso~~althin~Iria in some animals without exception?. At any rate, further studies are needed for the elucidation of the mechanism of isovalthine production in hypercholesterolemic patients. ACKNOWLEDGEMENT
The continued interest of Prof. Dr. S. Mizuhara in this study is gratefully acknowledged. REFERENCES AND S. OHMORI, Arch. BiocAem. Biophvs., gz (1961) 53. AND S. MTZUHARA, A&. B~~che~~.B~o~k~s., 96 (1~62) 179. Arch. Biochem. Bioph_vs., 104 (1964) 509. R. G. WESTALL, B&hem. J.. 55 (1955) 244. I. Z.%RIN AND K. BLOCK, ,J, Biol. Chem., rgz (1951) 207 P. V. AVIZONIS AND J. C. WRISTON, Biochim. Biophys. Acta, 34 (1956) 279. I<. FUKUTOME, Acta Med. Ok~.yama, 16 (rgGz) 241. ;47. T. UBUKA, _4cta Med. Okuyama, 17 (1963) 273. T. TJBuK.~,K. HORIUCHI, T. SHIMOMUR~ AND S. MlzuH~R~,Acta~ed. Okayama, r8(rg64) 65. 0. W. PORTMAN AND G. V.MANN,J. Bid. Chem., 213 (1955) 733. J. C. SEIDEL AND A. E. HARPER, Proc. Sot. Exptl. Biol. Med., III (1962) 579. T. KUW~KI, S. OHMORI AND S. MIZUHARA, Biochim. Biophys. Acta, 78 (x963) 553. T. K~~AKI, J. Biochem., 57 (1965) 125. T. UBUKA, J. B&hem.. 52 (1962) 440. G. M. TOMKINS, H. SHEPPARD AND I. L. CHAIKOFF,J.B~OZ. Chem., 203 (1953) 781. D. STEINBERG AND D.S. FREDRICKSON, Al8n.N.Y. Acad.Sci.,64(rg56) 579. W. T. BEHER, G. D. BAKER AND W. L. ANTHONY, Proc. Sot. Exptl. Biol. Med., rog (rg6z) 863.
I S.MIZUHARA 2 S. OHMORI 3 S. OHMORI,
4 i
7 8 g
IO I T 12 13 14 15
16 77
Cl&z. Chim.
Acta,
12 (1965) 325-329