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The antithyrotoxic factor: Its splubilization and relation to intestinal xanthine oxidase
The Antithyrotoxic Factor : Its Solubilization and Relation to Intestinal Xanthine Oxidase’ Boyd L. O’Dell, Sidney J. Stolzenberg,z Joseph H. Bruemmer and Albert G. Hogan From the Department of Agricultural Missouri, Columbia, Received
July
Chemistry, Missouri
University
of
12, 1954
Several of the vitamins have been shown to be antithyrotoxic inasmuch as they tend to counteract the growth-retarding effect of thyroxine when it is added to an otherwise adequate diet. This effect is usually ascribed to the fact that the stress of hyperthyroidism increases the requirement for these vitamins. The term antithyrotoxic factor (ATF) has been widely used to describe an unrecognized factor that occurs in liver (1, 2), soybean oil meal (3), and other natural products (4). This factor counteracts growth retardation and increases survival of hyperthyroid rats fed a casein-type diet containing the known vitamins required by the rat. Vitamin BH counteracts thyrotoxicity in rats fed a diet containing soybean protein (5, 6), but it is not identical with the ATF (7). The chick and the hyperthyroid rat require an unrecognized factor that can be extracted from dextrin with 70 % ethanol, but which occurs more abundantly in liver and primary yeast (8). The factor was stable to heat and alkali treatment, was not adsorbed on activated carbon (Norit), and was not dialyzable. The distribution of the ATF is similar to that of the xanthine oxidase factor (9), but concentrates of this factor did not exert any antithyrotoxic effect. More recently the xanthine oxidase factor has been identified as molybdenum (lo-12), and xanthine oxidase has been shown to contain molybdenum (12-15) as well as riboflavin. 1 Contribution from the Missouri Agricultural Experiment Series No. 1447. 2 Present address: Lederle Laboratories Division, American River, New York. 232
Station, Cyanamid,
Journal Pearl
ANTITHYROTOXIC
FACTOR
AND XANTHINE
OXIDASE
233
In this report methods are presented for solubilizing the antithyrotoxic factor as it occurs in the water-insoluble protein of liver, and evidence is presented to show that, when rats consume a diet containing iodinated casein, liver protein increases the intestinal xanthine oxidase activity to saturation level, but molybdenum alone does not. EXPERIMENTAL
A. Animal Assay of the ATF Albino rats of the Wistar strain (stock obtained from Texas A & M College) were weaned at 3 weeks of age (35-45 g.) and fed a basal ration that contained 0.15% of iodinated casein for a l-week depletion period. They were then divided into groups so as to obtain an equal distribution of animals of the same litter, weight, and sex. For each assay, approximately equal numbers of animals received the basal ration, a positive control ration that contained 10% of liver residue4 and a ration that contained the supplement under test. Feed and water were supplied ad Zibitl~n~ for 2 weeks, and the gain in weight was recorded. The supplements were added at the expense of casein and cerelose so as to maintain a constant percentage of dietary nitrogen. The composition of the basal rat,ion was: water-washed casein 22 g.; cerelose 65 g.; wood pulp 3 g.; salts (16) 5 g.; lard 5 g.; thiamine hydrochloride5 7.0 mg.; riboflavin 1.6 mg.; pyridoxine hydrochloride 1.6 mg.; Ca pantothenate 6.0 mg.; biotin 0.02 mg.; choline chloride 100 mg.; folacin6 0.5 mg.; inositol 50.0 mg.; p-aminobenzoic acid 5.0 mg.; niacin 5.0 mg.; vitamin Blz 5.0 pg.; vitamin A 2000 I.U.; vitamin D 280 I.U.; a-tocopherol3.0 mg.; menadione 1.0 mg.; and iodinated casein 0.15 g. In some of the trials in which xanthine oxidase was determined, the iodinated casein wa,s omitted, and in some cases molybdenum was added in the form of sodium molybdate.
B. Xanthine
Oxidase Assay
Intestinal xanthine oxidase was determined by the method of Axelrod and Elvehjem (17) as used by Westerfeld and Richert (18). Approximately 20 cm. of the upper intestine was homogenized with 9 vol. of sodium and potassium phosphate buffer (pH 7.4), and 2.7 ml. of this homogenate was placed in the main compartment,s of each of t%-o Warburg flasks. The side arm of one flask contained 0.3 ml. of water and the other 0.3 ml. of 0.03 M xanthine. The endogenous respiration was partially exhausted by incubation at 38°C. for 1 hr. before the substrate was tipped in. Calculations were based on the difference in oxygen consumption during 3 Protamone, Cerophyll Laboratories, Inc., obtained C. W. Turner, University of Missouri. 4 The heat-coagulated protein of beef liver which has extraction. This product was generously supplied by Chicago, Illinois. 6 The folacin was generously donated by the Lederle American Cyanamid, Pearl River, New York, and the mins by Merck and Co. Rahway, New Jersey.
through
the courtesy
of
Dr.
been defatted by solvent Armour and Company, Laboratories Division of other water-soluble vita-
234
O’DELL ET AL.
three lo-min. periods, and xanthine oxidase activity is expressed as the microliters of oxygen consumed per hour by 270 mg. of fresh tissue.
C. Methods of Solutilizing Enzymatic Hydrolysis. The pepsin digest was prepared by incubating 1 kg. of defatted liver residue at 38°C. with 50 g. of pepsin powder (N.F.). The residue was suspended in 20 1. of water, adjusted to pH 2.0, and readjusted twice daily during a I-day period. Finally the solution was adjusted to pH 5 and extracted with hot water. The dry matter in the extract amounted to approximately 50% of the original residue (corrected for the salt added). The pancreatin and trypsin digests were prepared in an analogous manner, using commercial pancreatin (U.&P.) and 300-l. trypsin, respectively. The suspensions were adjusted to pH 8.0 during the digestion. The dry matter yield in the pancreatin extract was 70% and in the trypsin extract, 50% (corrected for the salt added). Alkali and Acid Hydrolysis. One kilogram of defatted liver residue was autoclaved in 10 1. of 0.1 N sodium hydroxide at 15 lb. pressure for 4 hr., and the soluble portion was adjusted to pH 7.0. The yield of dry matter was 80% (corrected for the salt added). One kilogram of defatted liver residue was suspended in 10 1. of 1 N sulfuric acid and autoclaved for 18 hr. The soluble portion was adjusted to pH 4.5 with barium hydroxide, and the barium sulfate was removed. The dry matter yield was 70%. The hydrolysis may be performed by refluxing for 48 hr., but the yield is decreased.
RESULTS
A. Sources OJ the Antithyrotoxic
Factor and Methods of Solubilizing
The results obtained in assays of source materials for the ATF are shown in Table I. Summaries of all animals fed the basal and positive control rations are given in the first, two lines. The average gain in 2 weeks of 121 animals fed 10 $& liver residue (positive controls) was 61 g. An aqueous extract of liver possessed an appreciable amount of activity, but it, was less active than the insoluble residue. Brewer’s yeast was inactive in the assay, and wheat germ and soybean oil meal were only moderately active. A hot-water extract of soybean oil meal fed at a level of 5 % was inactive, but 25 % of the insoluble residue was at, least as active as 10 y0 of liver residue. When 10 $ZOof gelatin replaced an equal amount of casein in the basal ration, the rate of growth was not, improved. If gelatin contains the antithyrotoxic factor, its effect, was overshadowed by the poor quality of protein which resulted from the substitution of gelatin for casein. The results obtained with the various hydrolytic reagents are shown in Table II. All of the proteolytic enzymes tested released the anti-
ANTITHYROTOXIC
FACTOR
AND
TABLE
XANTHINE
I
Sources of the Antithyrotoxic
Factor
Supplement
Gain
Description
Animals
70 None Defatted liver residue Aqueous extract of liver Brewer’s yeast Wheat germ Soybean oil meal (SBOM) Aqueous extract of SBOM Insoluble residue of SBOM Gelatin
Supplement
.-
10 5 25 10
10
10
I
52 f 47 f 54f 54f 55f 63f 39f
in weight,
2 weeks Positive controla
Basal
g.
NO.
121b 1416 8 8 7 8 6 8
10 10 10
235
OXIDASE
g.
g.
12 10 6 9 7 7 6
45 f
101
46 f 45 f 49 f 48 f 54% 34f 41f
11 12 10
10 9 7 6
61 f 61 f 63 f 66 f 66 f 69% 60f 64f
10 10 11 10 10 8 6 8
e The positive control group received 10% of liver residue. b These numbers represent all the animals that received the basal and positive control rations. c Standard deviation.
thyrotoxic factor, but the mixture found in pancreatin was most effective both from the standpoint of yield and activity. However, when the pancreatin and pepsin digests were fed at levels of 5 %, the response was not equivalent to the positive control. The trypsin digest at a level of 10 y0 was also inferior to the positive control. There was a depression of growth and evidence of diarrhea when the supplements were fed at a level of 10%. This may have been the result of the salts introduced during the pH adjustments. The sodium hydroxide and sulfuric acid hydrolyzates were as effective as the original liver residue. Hydrolysis with sulfuric acid has the advantage that the salt can be removed and the hydrolysis is sufficiently complete that the material is soluble at all pH values. The sodium hydroxide-treated material was completely soluble only at pH 7 or above. The portion soluble at pH 5.0 was virtually inactive. B. Miscellaneous Substances Tested for ATF Activity Various known compounds have been tested with entirely negative results. Although lyxoflavin6 has been reported to stimulate the growth E The lyxoflavin Company, Rahway,
was generously New Jersey.
supplied
by Dr.
Karl
Folkers,
Merck
and
236
O’DELL
Methods
of Solubilizing
ET
AL.
TABLE II the Antithyrotoxic
Factor
Supplement
in Liver
Residue
Gain in weight, 2 weeks --t-
Description
Pepsin digest Pancreatin digest Trypsin digest 0.1 N NaOH hydrolyzate 1 N H2S04 hydrolyzate
Amount
Animals
%
NO.
5 5 10 10 10
6 10 12 10 13
a These gains are significantly higher the basal ration as shown by the Fisher
Supplement g.
56 59 51 62f 59
g.
f 10 f 80 III 75 7a f 6”
(at the 5yo level
yiBi;g?
Basal
49 52 41 53 40
f f f f f
g.
5 7 6 8 5
63 64 60 62 59
f f f f f
5 7 8 3 8
or less) than those on
t test.
of rats fed a ration containing soybean meal and thyroid powder (19), it was inactive in this assay when fed at a level of 1.5 mg./lOO g. diet. Thioctic acid’ (0.1 mg. %) erotic acid (2 mg. sl,) and guanine (0.1%) were also inactive as previously reported (21). A mixture of glycine (1 Yo), DL-methionine (0.5 %), and L-arginine (0.5 Cj&) supported no better rate of gain than the basal ration. The growth of the guinea pig is stimulated by feeding more potassium and magnesium than are usually fed to the rat (22), but a combination of potassium acetate (2.5%) and magnesium oxide (0.5 %) did not stimulate the growth of the hyperthyroid rat. C. Intestinal
Xanthine Oxidase Activity in the Hyperthyroid
Rat
Supplementation of a purified casein diet with molybdenum has been reported to increase the xanthine oxidase activity of rat intestine to values comparable to those obtained with liver residue (10, 11). This observation has been confirmed in this study (see col. 3, Table III.). When the rats received the same basal ration with iodinated casein added (col. 4), the intestinal xanthine oxidase activity was the same as without the thyroactive substance. This is also in agreement with reported observations (23), but the addition of molybdenum (0.4-1.6 mg./kg.) to the iodinated casein basal diet did not give maximal xanthine oxidase values. On the other hand, liver residue gave values comparable to those observed on the diet without the thyroxine added. 7 The DL-6-thioctic acid was generously supplied by Dr. T. H. Jukes, Laboratories, American Cyanamid, Pearl River, New York.
Lederle
ANTITHYROTOXIC
FACTOR
TABLE
Relation of the Antithyrotoxic
AND
XANTHINE
III
Factor and Intestinal
Xanthine oxidase activity” No. of animals per group
Supplement
237
OXIDASE
Iod. casein suppl.” NOM
O.ls%
Xanthine
Oxidase
Weight gain, 2 weeks Iod. casein supp1.b NOW
0.15%
__PZ. 0%
None Mot L. R.d
I
9 18 8
9f 79 * 73 f
8 12 19
/Al. 02
8f 7 49 zk m 73 f 15
g.
74 * 65z.t 69 f
c.
12 8 10
42f 45* 58 f
7 7 10
a The xanthine oxidase activity is expressed as the microliters of oxygen uptake per hour per 270 mg. of fresh tissue. The standard deviations are indicated. b The iodinated casein was “protamone.” c The molybdenum supplement varied from 1 to 4 mg. of sodium molybdate/ kg. ration. Since there was no difference in response, the data were combined. d Liver residue was added at a level of 10%. E This value is statistically different at the 1% level from the group that received liver residue and iodinated casein as well as from the group that received molybdenum and no iodinated casein.
Liver residue (Armour’s non-defatted) contains 1.8 pg. molybdenum/g. (personal communication from Dr. D. A. Richert). Therefore, 10% of defatted liver residue would supply about 240 pg./kg. ration. The addition of molybdenum to the basal ration containing iodinated casein gave intestinal xanthine oxidase values which were two-thirds of those obtained with liver residue. Although molybdenum stimulated xanthine oxidase activity, it had no effect on growth. On the other hand, the addition of liver residue to the iodinated casein diet gave maximal intestinal xanthine oxidase activity and produced a growth stimulation of about 30%. However, liver residue did not support a rate of gain equivalent to that observed on the thyroxine-free basal ration. Thus, it appears that liver residue contains a factor(s) which is required to give maximal xanthine oxidase activity and to stimulate growth only in the case of the hyperthyroid rat. In the presence of adequate molybdenum there is a close correlation between the rate of gain of the animals and the amount of intestinal xanthine oxidase activity. As may be observed in Table III, the gain on the molybdenum diet was 45 g. and the xanthine oxidase activity was 49 pl., whereas on the liver residue diet the gain was 58 g. and the xanthine oxidase activity was 73 ~1.
238
O'DELL ET AL.
DISCUSSION The ATF as it occurs in soybean oil meal as well as in liver tissue is relatively insoluble in water, but it can be released by various reagents that hydrolyze the peptide bond. From a preparative standpoint, sulfuric acid hydrolysis seems to be the most promising in that it is possible to obtain relatively good yields of soluble material without introducing large amounts of salts. The fact that the factor is released by proteolytic reagents indicates that it is either amino acid or peptide in nature or that it is bound to protein. Since all the sources of the ATF used are relatively high in protein, the possibility remains that it is an amino acid or combination of amino acids. The factor in liver residue that stimulates the growth of the hyperthyroid rat may be identical with the factor that stimulates the xanthine oxidase activity, but proof of their identity must await isolation and/or identification. The concentration of xanthine oxidase in rat intestine is affected by dietary riboflavin, molybdenum, and protein, but liver xanthine oxidase depends primarily on the protein content of the diet (24). A series of investigations reported by the Wisconsin group (25, 26) indicate that liver xanthine oxidase is a sensitive index to subtle changes in dietary protein and that there is close correlation between the enzyme activity and growth of rats fed proteins of different biological value. In the case of the hyperthyroid rat fed a casein diet supplemented with molybdenum, there is also a correlation between the rate of growth and the intestinal xanthine oxidase activity when a portion of the casein is replaced by liver residue. It is possible that the liver residue supplies a more favorable amino acid balance than the casein alone, and this deficiency of casein is not observed in the normal animal. On the other hand, liver residue may supply an unrecognized factor required for the optimum synthesis or activity of the enzyme. SUMMARY
1. The major portion of the antithyrotoxic factor in liver and soybean meal is not extractable with water, but the factor becomes water soluble upon hydrolysis with proteolytic enzymes, sodium hydroxide, or sulfuric acid. 2. Although molybdenum produces the saturation level of intestinal xanthine oxidase activity in normal rats fed a casein-type diet, in the hyperthyroid rat it gives values of about two-thirds of those obtained with liver residue.
ANTITHYROTOXIC
FACTOR AND XANTHINE
OXIDASE
239
3. The correlation between the growth of rats fed iodinated casein and the level of intestinal xanthine oxidase activity suggests that the antithyrotoxic factor may be required to give maximum xanthine oxidase activity when the animal is under stress. REFERENCES 1. ERSHOFF, B. H., Proc. Sot. Exptl. Biol. Med. 64, 500 (1947). 2. BETHEIL, J. J., WIEBELHAUS, V. D., AND LARDY, H. A., J. Nutrition
34, 413
(1947). 3. ERSHOFF, B. H., J. Nutrition 39, 259 (1949). 4. TAPPAN, D. V., BOLDT, R. E., AND ELVEHJEM, C. A., Proc. Sot. Exptl. Biol. Med. 63, 135 (1953). 5. REGISTER, U. D., RUEGAMER, W. R., AND ELVEHJEM, C. A., J. Biol. Chem. 177, 129 (1949). 6. EMERSON, G. A., Proc. Sot. Exptl. Biol. Med. 70, 392 (1949). 7. ERSHOFF, B. H., Proc. Sot. Exptl. Biol. Med. 71, 209 (1949). 8. DIETRICH, L. S., MONSON, W. J., AND ELVEHJEM, C. A., Arch. Biochem. and Biophys. 36, 91 (1952). 9. RICHERT, D. A., AND WESTERFELD, W. W., J. Biol. Chem. 19!& 49 (1951). 10. DERENZO, E. C., KALEITA, E., HEYTLER, P. G., OLESON, J. J., HUTCHINGS, B. L., AND WILLIAMS, J. H., J. Am. Chem. Sot. 76, 753 (1953). 11. DERENZO, E. C., KALEITA, E., HEYTLER, P. G., OLESON, J. J., HUT~XINGS, B. L., AND WILLIAMS, J. H., Arch. Biochem. and Biophys. 46, 247 (1953). 12. RICRERT, D. A., AND WESTERFELD, W. W., J. Biol. Chem. 203, 915 (1953). 13. TOTTER, J. R., BURNETT, W. T., JR., MONROE, R. A., WHITNEY, I. B., AND COMAR, C. L., Science 118, 555 (1953). 14. GREEN, D. E., AND BEINERT, H., Biochim. et Biophys. Acta 11, 599 (1953). 15. DERENZO, E. C., HEYTLER, P. G., AND KALEITA, E., Arch. Biochem. and Biophys. 49, 242 (1954). 16. RICHARDSON, L. R., AND HOGAN, A. G., J. Nutrition 32,459 (1946). 17. AXELROD, A. E., AND ELVEHJEM, C. A., J. Biol Chem. 140,725 (1941). 18. WES~ERFELD, W. W., AND RICHERT, D. A., J. Biol. Chem. 192, 35 (1951). 19. EMERSON, G. A., AND FOLKERS, K., J. Am. Chem. Sot. 73,2398 (1951). 20. ERSHOFF, B. H., Proc. Sot. Expfl. BioZ. Med. 79, 469 (1952). 21. OVERBY, L. R., FREDERICKSON, R. L., AND FROST, D. V., Federation Proc. 12, 425 (1953). 22. ROINE, P., BOOTH, A. N., ELVEHJEM, C. A., AND HART, E. B., Proc. Sot. Exptl. Biol. Med. 71, 90 (1949). 23. WESTERFELD, W. W., AND RICHERT, D. A., J. Biol. Chem. 199, 819 (1952). 24. RICHERT, D. A., AND WESTERFELD, W. W., Proc. Sot. Exptl. Biol. Med. 63, 726 (1953). 25. WILLIAMS, J. N., JR., AND ELVEHJEM, C. A., J. Biol. Chem. 181, 559 (1949). 26. LITWACK, G., WILLIAMS, J. N., JR., FATTERPAKER, P., CHEN, L., AND ELVEHJEM, C. A., J. Nutrition 49, 579 (1953).
July
Chemistry, Missouri
University
of
12, 1954
Several of the vitamins have been shown to be antithyrotoxic inasmuch as they tend to counteract the growth-retarding effect of thyroxine when it is added to an otherwise adequate diet. This effect is usually ascribed to the fact that the stress of hyperthyroidism increases the requirement for these vitamins. The term antithyrotoxic factor (ATF) has been widely used to describe an unrecognized factor that occurs in liver (1, 2), soybean oil meal (3), and other natural products (4). This factor counteracts growth retardation and increases survival of hyperthyroid rats fed a casein-type diet containing the known vitamins required by the rat. Vitamin BH counteracts thyrotoxicity in rats fed a diet containing soybean protein (5, 6), but it is not identical with the ATF (7). The chick and the hyperthyroid rat require an unrecognized factor that can be extracted from dextrin with 70 % ethanol, but which occurs more abundantly in liver and primary yeast (8). The factor was stable to heat and alkali treatment, was not adsorbed on activated carbon (Norit), and was not dialyzable. The distribution of the ATF is similar to that of the xanthine oxidase factor (9), but concentrates of this factor did not exert any antithyrotoxic effect. More recently the xanthine oxidase factor has been identified as molybdenum (lo-12), and xanthine oxidase has been shown to contain molybdenum (12-15) as well as riboflavin. 1 Contribution from the Missouri Agricultural Experiment Series No. 1447. 2 Present address: Lederle Laboratories Division, American River, New York. 232
Station, Cyanamid,
Journal Pearl
ANTITHYROTOXIC
FACTOR
AND XANTHINE
OXIDASE
233
In this report methods are presented for solubilizing the antithyrotoxic factor as it occurs in the water-insoluble protein of liver, and evidence is presented to show that, when rats consume a diet containing iodinated casein, liver protein increases the intestinal xanthine oxidase activity to saturation level, but molybdenum alone does not. EXPERIMENTAL
A. Animal Assay of the ATF Albino rats of the Wistar strain (stock obtained from Texas A & M College) were weaned at 3 weeks of age (35-45 g.) and fed a basal ration that contained 0.15% of iodinated casein for a l-week depletion period. They were then divided into groups so as to obtain an equal distribution of animals of the same litter, weight, and sex. For each assay, approximately equal numbers of animals received the basal ration, a positive control ration that contained 10% of liver residue4 and a ration that contained the supplement under test. Feed and water were supplied ad Zibitl~n~ for 2 weeks, and the gain in weight was recorded. The supplements were added at the expense of casein and cerelose so as to maintain a constant percentage of dietary nitrogen. The composition of the basal rat,ion was: water-washed casein 22 g.; cerelose 65 g.; wood pulp 3 g.; salts (16) 5 g.; lard 5 g.; thiamine hydrochloride5 7.0 mg.; riboflavin 1.6 mg.; pyridoxine hydrochloride 1.6 mg.; Ca pantothenate 6.0 mg.; biotin 0.02 mg.; choline chloride 100 mg.; folacin6 0.5 mg.; inositol 50.0 mg.; p-aminobenzoic acid 5.0 mg.; niacin 5.0 mg.; vitamin Blz 5.0 pg.; vitamin A 2000 I.U.; vitamin D 280 I.U.; a-tocopherol3.0 mg.; menadione 1.0 mg.; and iodinated casein 0.15 g. In some of the trials in which xanthine oxidase was determined, the iodinated casein wa,s omitted, and in some cases molybdenum was added in the form of sodium molybdate.
B. Xanthine
Oxidase Assay
Intestinal xanthine oxidase was determined by the method of Axelrod and Elvehjem (17) as used by Westerfeld and Richert (18). Approximately 20 cm. of the upper intestine was homogenized with 9 vol. of sodium and potassium phosphate buffer (pH 7.4), and 2.7 ml. of this homogenate was placed in the main compartment,s of each of t%-o Warburg flasks. The side arm of one flask contained 0.3 ml. of water and the other 0.3 ml. of 0.03 M xanthine. The endogenous respiration was partially exhausted by incubation at 38°C. for 1 hr. before the substrate was tipped in. Calculations were based on the difference in oxygen consumption during 3 Protamone, Cerophyll Laboratories, Inc., obtained C. W. Turner, University of Missouri. 4 The heat-coagulated protein of beef liver which has extraction. This product was generously supplied by Chicago, Illinois. 6 The folacin was generously donated by the Lederle American Cyanamid, Pearl River, New York, and the mins by Merck and Co. Rahway, New Jersey.
through
the courtesy
of
Dr.
been defatted by solvent Armour and Company, Laboratories Division of other water-soluble vita-
234
O’DELL ET AL.
three lo-min. periods, and xanthine oxidase activity is expressed as the microliters of oxygen consumed per hour by 270 mg. of fresh tissue.
C. Methods of Solutilizing Enzymatic Hydrolysis. The pepsin digest was prepared by incubating 1 kg. of defatted liver residue at 38°C. with 50 g. of pepsin powder (N.F.). The residue was suspended in 20 1. of water, adjusted to pH 2.0, and readjusted twice daily during a I-day period. Finally the solution was adjusted to pH 5 and extracted with hot water. The dry matter in the extract amounted to approximately 50% of the original residue (corrected for the salt added). The pancreatin and trypsin digests were prepared in an analogous manner, using commercial pancreatin (U.&P.) and 300-l. trypsin, respectively. The suspensions were adjusted to pH 8.0 during the digestion. The dry matter yield in the pancreatin extract was 70% and in the trypsin extract, 50% (corrected for the salt added). Alkali and Acid Hydrolysis. One kilogram of defatted liver residue was autoclaved in 10 1. of 0.1 N sodium hydroxide at 15 lb. pressure for 4 hr., and the soluble portion was adjusted to pH 7.0. The yield of dry matter was 80% (corrected for the salt added). One kilogram of defatted liver residue was suspended in 10 1. of 1 N sulfuric acid and autoclaved for 18 hr. The soluble portion was adjusted to pH 4.5 with barium hydroxide, and the barium sulfate was removed. The dry matter yield was 70%. The hydrolysis may be performed by refluxing for 48 hr., but the yield is decreased.
RESULTS
A. Sources OJ the Antithyrotoxic
Factor and Methods of Solubilizing
The results obtained in assays of source materials for the ATF are shown in Table I. Summaries of all animals fed the basal and positive control rations are given in the first, two lines. The average gain in 2 weeks of 121 animals fed 10 $& liver residue (positive controls) was 61 g. An aqueous extract of liver possessed an appreciable amount of activity, but it, was less active than the insoluble residue. Brewer’s yeast was inactive in the assay, and wheat germ and soybean oil meal were only moderately active. A hot-water extract of soybean oil meal fed at a level of 5 % was inactive, but 25 % of the insoluble residue was at, least as active as 10 y0 of liver residue. When 10 $ZOof gelatin replaced an equal amount of casein in the basal ration, the rate of growth was not, improved. If gelatin contains the antithyrotoxic factor, its effect, was overshadowed by the poor quality of protein which resulted from the substitution of gelatin for casein. The results obtained with the various hydrolytic reagents are shown in Table II. All of the proteolytic enzymes tested released the anti-
ANTITHYROTOXIC
FACTOR
AND
TABLE
XANTHINE
I
Sources of the Antithyrotoxic
Factor
Supplement
Gain
Description
Animals
70 None Defatted liver residue Aqueous extract of liver Brewer’s yeast Wheat germ Soybean oil meal (SBOM) Aqueous extract of SBOM Insoluble residue of SBOM Gelatin
Supplement
.-
10 5 25 10
10
10
I
52 f 47 f 54f 54f 55f 63f 39f
in weight,
2 weeks Positive controla
Basal
g.
NO.
121b 1416 8 8 7 8 6 8
10 10 10
235
OXIDASE
g.
g.
12 10 6 9 7 7 6
45 f
101
46 f 45 f 49 f 48 f 54% 34f 41f
11 12 10
10 9 7 6
61 f 61 f 63 f 66 f 66 f 69% 60f 64f
10 10 11 10 10 8 6 8
e The positive control group received 10% of liver residue. b These numbers represent all the animals that received the basal and positive control rations. c Standard deviation.
thyrotoxic factor, but the mixture found in pancreatin was most effective both from the standpoint of yield and activity. However, when the pancreatin and pepsin digests were fed at levels of 5 %, the response was not equivalent to the positive control. The trypsin digest at a level of 10 y0 was also inferior to the positive control. There was a depression of growth and evidence of diarrhea when the supplements were fed at a level of 10%. This may have been the result of the salts introduced during the pH adjustments. The sodium hydroxide and sulfuric acid hydrolyzates were as effective as the original liver residue. Hydrolysis with sulfuric acid has the advantage that the salt can be removed and the hydrolysis is sufficiently complete that the material is soluble at all pH values. The sodium hydroxide-treated material was completely soluble only at pH 7 or above. The portion soluble at pH 5.0 was virtually inactive. B. Miscellaneous Substances Tested for ATF Activity Various known compounds have been tested with entirely negative results. Although lyxoflavin6 has been reported to stimulate the growth E The lyxoflavin Company, Rahway,
was generously New Jersey.
supplied
by Dr.
Karl
Folkers,
Merck
and
236
O’DELL
Methods
of Solubilizing
ET
AL.
TABLE II the Antithyrotoxic
Factor
Supplement
in Liver
Residue
Gain in weight, 2 weeks --t-
Description
Pepsin digest Pancreatin digest Trypsin digest 0.1 N NaOH hydrolyzate 1 N H2S04 hydrolyzate
Amount
Animals
%
NO.
5 5 10 10 10
6 10 12 10 13
a These gains are significantly higher the basal ration as shown by the Fisher
Supplement g.
56 59 51 62f 59
g.
f 10 f 80 III 75 7a f 6”
(at the 5yo level
yiBi;g?
Basal
49 52 41 53 40
f f f f f
g.
5 7 6 8 5
63 64 60 62 59
f f f f f
5 7 8 3 8
or less) than those on
t test.
of rats fed a ration containing soybean meal and thyroid powder (19), it was inactive in this assay when fed at a level of 1.5 mg./lOO g. diet. Thioctic acid’ (0.1 mg. %) erotic acid (2 mg. sl,) and guanine (0.1%) were also inactive as previously reported (21). A mixture of glycine (1 Yo), DL-methionine (0.5 %), and L-arginine (0.5 Cj&) supported no better rate of gain than the basal ration. The growth of the guinea pig is stimulated by feeding more potassium and magnesium than are usually fed to the rat (22), but a combination of potassium acetate (2.5%) and magnesium oxide (0.5 %) did not stimulate the growth of the hyperthyroid rat. C. Intestinal
Xanthine Oxidase Activity in the Hyperthyroid
Rat
Supplementation of a purified casein diet with molybdenum has been reported to increase the xanthine oxidase activity of rat intestine to values comparable to those obtained with liver residue (10, 11). This observation has been confirmed in this study (see col. 3, Table III.). When the rats received the same basal ration with iodinated casein added (col. 4), the intestinal xanthine oxidase activity was the same as without the thyroactive substance. This is also in agreement with reported observations (23), but the addition of molybdenum (0.4-1.6 mg./kg.) to the iodinated casein basal diet did not give maximal xanthine oxidase values. On the other hand, liver residue gave values comparable to those observed on the diet without the thyroxine added. 7 The DL-6-thioctic acid was generously supplied by Dr. T. H. Jukes, Laboratories, American Cyanamid, Pearl River, New York.
Lederle
ANTITHYROTOXIC
FACTOR
TABLE
Relation of the Antithyrotoxic
AND
XANTHINE
III
Factor and Intestinal
Xanthine oxidase activity” No. of animals per group
Supplement
237
OXIDASE
Iod. casein suppl.” NOM
O.ls%
Xanthine
Oxidase
Weight gain, 2 weeks Iod. casein supp1.b NOW
0.15%
__PZ. 0%
None Mot L. R.d
I
9 18 8
9f 79 * 73 f
8 12 19
/Al. 02
8f 7 49 zk m 73 f 15
g.
74 * 65z.t 69 f
c.
12 8 10
42f 45* 58 f
7 7 10
a The xanthine oxidase activity is expressed as the microliters of oxygen uptake per hour per 270 mg. of fresh tissue. The standard deviations are indicated. b The iodinated casein was “protamone.” c The molybdenum supplement varied from 1 to 4 mg. of sodium molybdate/ kg. ration. Since there was no difference in response, the data were combined. d Liver residue was added at a level of 10%. E This value is statistically different at the 1% level from the group that received liver residue and iodinated casein as well as from the group that received molybdenum and no iodinated casein.
Liver residue (Armour’s non-defatted) contains 1.8 pg. molybdenum/g. (personal communication from Dr. D. A. Richert). Therefore, 10% of defatted liver residue would supply about 240 pg./kg. ration. The addition of molybdenum to the basal ration containing iodinated casein gave intestinal xanthine oxidase values which were two-thirds of those obtained with liver residue. Although molybdenum stimulated xanthine oxidase activity, it had no effect on growth. On the other hand, the addition of liver residue to the iodinated casein diet gave maximal intestinal xanthine oxidase activity and produced a growth stimulation of about 30%. However, liver residue did not support a rate of gain equivalent to that observed on the thyroxine-free basal ration. Thus, it appears that liver residue contains a factor(s) which is required to give maximal xanthine oxidase activity and to stimulate growth only in the case of the hyperthyroid rat. In the presence of adequate molybdenum there is a close correlation between the rate of gain of the animals and the amount of intestinal xanthine oxidase activity. As may be observed in Table III, the gain on the molybdenum diet was 45 g. and the xanthine oxidase activity was 49 pl., whereas on the liver residue diet the gain was 58 g. and the xanthine oxidase activity was 73 ~1.
238
O'DELL ET AL.
DISCUSSION The ATF as it occurs in soybean oil meal as well as in liver tissue is relatively insoluble in water, but it can be released by various reagents that hydrolyze the peptide bond. From a preparative standpoint, sulfuric acid hydrolysis seems to be the most promising in that it is possible to obtain relatively good yields of soluble material without introducing large amounts of salts. The fact that the factor is released by proteolytic reagents indicates that it is either amino acid or peptide in nature or that it is bound to protein. Since all the sources of the ATF used are relatively high in protein, the possibility remains that it is an amino acid or combination of amino acids. The factor in liver residue that stimulates the growth of the hyperthyroid rat may be identical with the factor that stimulates the xanthine oxidase activity, but proof of their identity must await isolation and/or identification. The concentration of xanthine oxidase in rat intestine is affected by dietary riboflavin, molybdenum, and protein, but liver xanthine oxidase depends primarily on the protein content of the diet (24). A series of investigations reported by the Wisconsin group (25, 26) indicate that liver xanthine oxidase is a sensitive index to subtle changes in dietary protein and that there is close correlation between the enzyme activity and growth of rats fed proteins of different biological value. In the case of the hyperthyroid rat fed a casein diet supplemented with molybdenum, there is also a correlation between the rate of growth and the intestinal xanthine oxidase activity when a portion of the casein is replaced by liver residue. It is possible that the liver residue supplies a more favorable amino acid balance than the casein alone, and this deficiency of casein is not observed in the normal animal. On the other hand, liver residue may supply an unrecognized factor required for the optimum synthesis or activity of the enzyme. SUMMARY
1. The major portion of the antithyrotoxic factor in liver and soybean meal is not extractable with water, but the factor becomes water soluble upon hydrolysis with proteolytic enzymes, sodium hydroxide, or sulfuric acid. 2. Although molybdenum produces the saturation level of intestinal xanthine oxidase activity in normal rats fed a casein-type diet, in the hyperthyroid rat it gives values of about two-thirds of those obtained with liver residue.
ANTITHYROTOXIC
FACTOR AND XANTHINE
OXIDASE
239
3. The correlation between the growth of rats fed iodinated casein and the level of intestinal xanthine oxidase activity suggests that the antithyrotoxic factor may be required to give maximum xanthine oxidase activity when the animal is under stress. REFERENCES 1. ERSHOFF, B. H., Proc. Sot. Exptl. Biol. Med. 64, 500 (1947). 2. BETHEIL, J. J., WIEBELHAUS, V. D., AND LARDY, H. A., J. Nutrition
34, 413
(1947). 3. ERSHOFF, B. H., J. Nutrition 39, 259 (1949). 4. TAPPAN, D. V., BOLDT, R. E., AND ELVEHJEM, C. A., Proc. Sot. Exptl. Biol. Med. 63, 135 (1953). 5. REGISTER, U. D., RUEGAMER, W. R., AND ELVEHJEM, C. A., J. Biol. Chem. 177, 129 (1949). 6. EMERSON, G. A., Proc. Sot. Exptl. Biol. Med. 70, 392 (1949). 7. ERSHOFF, B. H., Proc. Sot. Exptl. Biol. Med. 71, 209 (1949). 8. DIETRICH, L. S., MONSON, W. J., AND ELVEHJEM, C. A., Arch. Biochem. and Biophys. 36, 91 (1952). 9. RICHERT, D. A., AND WESTERFELD, W. W., J. Biol. Chem. 19!& 49 (1951). 10. DERENZO, E. C., KALEITA, E., HEYTLER, P. G., OLESON, J. J., HUTCHINGS, B. L., AND WILLIAMS, J. H., J. Am. Chem. Sot. 76, 753 (1953). 11. DERENZO, E. C., KALEITA, E., HEYTLER, P. G., OLESON, J. J., HUT~XINGS, B. L., AND WILLIAMS, J. H., Arch. Biochem. and Biophys. 46, 247 (1953). 12. RICRERT, D. A., AND WESTERFELD, W. W., J. Biol. Chem. 203, 915 (1953). 13. TOTTER, J. R., BURNETT, W. T., JR., MONROE, R. A., WHITNEY, I. B., AND COMAR, C. L., Science 118, 555 (1953). 14. GREEN, D. E., AND BEINERT, H., Biochim. et Biophys. Acta 11, 599 (1953). 15. DERENZO, E. C., HEYTLER, P. G., AND KALEITA, E., Arch. Biochem. and Biophys. 49, 242 (1954). 16. RICHARDSON, L. R., AND HOGAN, A. G., J. Nutrition 32,459 (1946). 17. AXELROD, A. E., AND ELVEHJEM, C. A., J. Biol Chem. 140,725 (1941). 18. WES~ERFELD, W. W., AND RICHERT, D. A., J. Biol. Chem. 192, 35 (1951). 19. EMERSON, G. A., AND FOLKERS, K., J. Am. Chem. Sot. 73,2398 (1951). 20. ERSHOFF, B. H., Proc. Sot. Expfl. BioZ. Med. 79, 469 (1952). 21. OVERBY, L. R., FREDERICKSON, R. L., AND FROST, D. V., Federation Proc. 12, 425 (1953). 22. ROINE, P., BOOTH, A. N., ELVEHJEM, C. A., AND HART, E. B., Proc. Sot. Exptl. Biol. Med. 71, 90 (1949). 23. WESTERFELD, W. W., AND RICHERT, D. A., J. Biol. Chem. 199, 819 (1952). 24. RICHERT, D. A., AND WESTERFELD, W. W., Proc. Sot. Exptl. Biol. Med. 63, 726 (1953). 25. WILLIAMS, J. N., JR., AND ELVEHJEM, C. A., J. Biol. Chem. 181, 559 (1949). 26. LITWACK, G., WILLIAMS, J. N., JR., FATTERPAKER, P., CHEN, L., AND ELVEHJEM, C. A., J. Nutrition 49, 579 (1953).