THE isolation and availability of crystalline

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1 Unidentified Factors in Poultry Nutrition. PROPERTIES AND PRELIMINARY FRACTIONATION OF A GROWTH FACTOR IN CONDENSED FISH SOLUBLES H. MENGE, C. A. DENTON, J. R. SIZEMORE, R. J. LILLIE AND H. R. BIRD Bureau of Animal Industry, United States Department of Agriculture, Beltsville, Maryland THE isolation and availability of crystalline vitamin B has stimulated the investigation of unidentified growth factors for poultry. This work has been comprehensively reviewed by Combs (9), Menge et al. (9a), and Briggs (9). Studies conducted in this laboratory have shown condensed fish solubles to contain an unidentified chick growth factor, or factors, Lillie et al. (9). The present investigation was undertaken to prepare a potent concentrate of this factor and to study some of its properties. EXPERIMENTAL PROCEDURE The method of assay used in the following studies has been described by Lillie et al. (9). The stability of the growth factor to autoclaving at various ph values was determined by adding either concentrated sodium hydroxide or concentrated hydrochloric acid to a sample of fish solubles diluted with an equal quantity of water. The total volume, after adjustment of ph, was kept uniform for each sample. Each lot was autoclaved for 0 minutes at pounds' pressure, cooled, neutralized, and mixed with the diet (Table, experiments and ). Centrifugation of fish solubles at,00 r.p.m. for hour resulted in the formation of three distinct layers. The topmost layer was discarded since it was found to be inactive in promoting chick growth. Centrifugation was used in subsequent work for the removal of these inert solids. A water extract of the two lower layers (Received for publication February 7, 9) 8 was obtained by adding 00 ml. of water and by centrifuging the mixture. The liquid phase was then decanted and the procedure was twice-repeated with the residue. The supernatants were then combined. An ashed sample of fish solubles was obtained by drying a weighed portion in vacuo and then subjecting the dried residue to a temperature of 0 C. in a muffle furnace for hours. The above fractions were fed in experiment (Table ). Alcohol extracts (70 percent methanol or 80 percent ethanol) were prepared in TABLE.- -Growth responses of chicks to prepared * t t t * G No UP Supplements L-lyxoflavin, mg./kg. L-lyxoflavin, 0 mg./kg. Essential amino acids+ Essential amino acids +, ph, autoclaved, ph, autoclaved, ph, autoclaved, ph, autoclaved, ph, autoclaved, ph 7, autoclaved, ph 9, autoclaved, ph, autoclaved S e? as =* * S SS S =* «* Av. wt. (gm.) weeks * Each group contained 0 male chicks per group at the start of lie experimental period (New Hampshire & XSilver Cornish 9). t Each group contained 0 New Hampshire male chicks at the start of the experimental period (Japanese method). X The following amino acids, expressed as percentage of total diet, were added: L-arginine HC, 0.0; L-histidine HC, 0.090; L-lysine HC, 0.08; L-leucine, 0.0; DL-isoleucine, 0.07; DL-valine, 0.08; DL-phenylaianine, 0.0; DLtryptophane, 0.0; glycine, 0.; L-glutamic acid, 0.; L- tyrosine, 0.0; DL-serine, 0.0; DL-methionine, 0.0; DLthreonine, 0.0; L-cystine, 0.0. Downloaded from at Penn State University (Paterno Lib) on May, 0

2 8 H. MENGE, C. A. DENTON, J. R. SIZEMORE, R. J. LILLIE AND H. R. BIRD TABLE. Chick growth responses to prepared AV, Wl Supplements (gm.) week f 9 S 9 Top layer (centrifugation) S Water extracts of lower layers * 8 Residue of water extracts a! Ashed fish solubles K 7t 97 e 0 80 EtOH sol., phenol layer S 0 80 EtOH sol., phenol layer?8 80 EtOH sol., water layer =* EtOH sol., water layer?8 0 8* S 80 EtOH sol., phenol layer S EtOH sol., phenol layer S EtOH sol., water layer * 8 80 EtOH sol., water layer e?8 0 * Experiment 8 contained 0 cross-bred male chicks (NH o" XSC 9) per group. t Experiments and 7 contained 0 New Hampshire male chicks (Japanese method) per group. the following manner: a weighed quantity of fish solubles was mixed with enough solvent to obtain the desired concentration allowing for the volume of water present in fish solubles (0 percent solids). The mixture was stirred for 0 minutes at room temperature and filtered with suction. The residue was reextracted twice with solvent of the desired concentration. The combined filtrates were concentrated and the organic solvents removed by distillation under reduced pressure. The residue remaining in the flask was taken up in water (experiments 7, 8 and 9). Two hundred milliliters of an 80 percent ethanol extract of fish solubles (equivalent to 00 gm. of original material) were used in the preparation of the phenol and water layer fractions (Table, experiments 7. and 8). One hundred twenty-five grams of crystalline phenol were heated with the alcohol fraction until dissolved. The mixture was then cooled to approximately room temperature and placed in a separatory funnel to allow the two layers to equilibrate. The water layer was reextracted with 0 grams of crystalline phenol in the same manner and the phenol layers from both extractions combined. The phenol was removed from each fraction by extraction with ether. A flow sheet showing the fractionation procedures which led to the most active fractions thus far obtained is given (Figure ). Each of these fractions was mixed directly into the basal diet at a level equivalent to either,, or 9 percent of the original fish solubles and fed in experiments 9 through. RESULTS AND DISCUSSION Bruins et al. (9) reported L-lyxoflavin to exhibit a growth response in chicks reared on a purified diet containing aureomycin and all other known required nutrients. However, lyxoflavin failed to elicit a response under the conditions of the present trial (Table, experiment ). These results indicate that lyxoflavin is not identical with the active principle in fish solubles. In experiments and (Table ), the essential amino acids were fed in quantities equivalent to those supplied by percent fish solubles. It is evident from the results of these two trials that the growth response obtained from fish solubles is not due to amino acids. The stability of the factor to autoclaving at a rather wide ph range is shown in experiments and (Table ). It appears that these treatments have little effect except at ph.0. The growth response obtained with the ph 9.0 fraction is below that which might be expected since apparently little of the growth activity was destoyed at a ph of 7.0 or.0. The growth responses obtained in experiment (Table ) indicate the active principle in fish solubles to be soluble in water. These data also show that centrifugation of the original sample is suitable for the removal of the inactive solids contained Downloaded from at Penn State University (Paterno Lib) on May, 0

3 GROWTH FACTOR IN FISH SOLUBLES 8 Pish solubles ( kg.). Centrifuged; top layer discarded. Dialyzed for days in liters of water at 0 - F.; water renewed every 8 hours Dialysis residue () Combined dialysates ( liters). Filtered through Whatman. Concentrated to,00 ml. in vacuo. Refrigerated over night; ppt. removed Precipitate (). Washed times with 00 ml. portions of cold 0 EtOH. Washings added to fraction (); residue discarded Dialysate (). Made to 0 EtOH by the addition of 9 EtOH; filtered in vacuo Supernatant (). Concentrated to,000 ml.; adjusted to ph.0. Equilibrated in separatory funnel with,000 ml. of phenol layer from phenol-water two-phase solubility system Phenol layer () Water layer (). Extract with ether; cone, to,000 ml.. Neutralize Precipitate (7) Water layer (0) Phenol layer () Water layer () Supernatant (8). Adjusted to ph.0; cone, to 70 ml.. Equilibrated in separatory funnel with 70 ml. of phenol layer from phenol-water two-phase solubility system Phenol layer (9). Extracted with ether; cone, to 00 ml.; adjusted to ph.0. Equilibrated in separatory funnel with ml. of the phenol layer and. ml. of the water layer of a two-phase phenol-water system plus. ml. of glacial acetic acid FIG.. Preparation of Fractions of Fish Solubles. Downloaded from at Penn State University (Paterno Lib) on May, 0 in the top layer. The lack of a growth response to fish solubles reduced to an ash indicates that the factor is not inorganic in nature, although this does not necessarily mean that a component of the active substance may not be inorganic. The solubility of the chick growth factor in 80 percent ethanol was established in experiments 7 and 8 (Table ). The results obtained in these trials also show the factor to be soluble to approximately the same extent in both layers of a phenolwater, two-phase solubility system. The growth responses obtained in

4 8 H. MENGE, C. A. DENTON, J. R. SIZEMORE, R. J. LILXIE AND H. R. BIRD TABLE. Growth responses of chicks to prepared 9* 0* * * t XT^P Supplements 70 MeOH insol. 70 MeOH sol. Dialysis residue () Dialysate () Dialysis residue () Dialysate () 0 EtOH insol. of dialysate 0 EtOH sol. of dialysate 0 EtOH insol. () 0 EtOH sol. () Phenol layer () Water layer (), ~ ~ =* ~ S? ~ ~ S ~ S ^ Av. wt. (gm.) weeks solids added _ * Experiment 8 through contained 0 cross-bred male chicks (N.H. cfxs.c. 9) per group. t Experiment contained 0 New Hampshire male chicks (Japanese method) per group. experiment 9 (Table ) show that the chick growth factor is soluble in 70 percent methanol and is dialyzable. It is also apparent from the results of experiments 8 through that the factor is soluble in 0 percent or 0 percent ethanol. Alcohol was used to precipitate a portion of the inert solids contained in the dialysate fraction. Fractions and represent the most potent concentrates of the fish solubles factor that we have obtained to date (Table ). On the basis of the total organic solids content of these fractions 9 to mg. per 00 grams of diet stimulated rapid growth of chicks. When compared with percent fish solubles, this represents an approximate concentration of thirty times. These data also demonstrate the suitability of the solvent system, phenol and water, for use in countercurrent distribution studies. The fractions given groups and (experiment ) represented samples prepared and assayed approximately months previous to this trial. Storage for the S-month period and removal of a precipitate which had formed apparently had little or no effect on the growth activity of the samples. No direct effort has been made to establish the identity of the factor contained in fish solubles with the growth factor reported to be present in liver by Menge et al. (9b). However, both the liver factor and the fish solubles factor appear to be dialyzable, stable to mild alkaline hydrolysis, soluble in 80 percent ethanol, phenol, and water, but insoluble in ether. This agreement has been attained even though widely different assay methods were used. These findings suggest that the two factors may be similar; on the other hand, differences in biological responses indicate that the substances are not identical (Lillie et al., 9). SUMMARY The fish solubles factor was shown to be soluble in water, phenol, 0, 0, and 80 percent ethanol, and 70 percent methanol, but insoluble in ether. The factor was also noted to be dialyzable and to be stable to autoclaving from ph.0 through.0, but not at ph.0. Prelimi- TABLE. Growth responses of chicks to prepared Group Supplements and Water layer () Phenol layer () Water layer ()* Phenol layer ()* Water layer (ll)t Phenol layer () t S S9 SL9 Av. wt. solids (gm.) added weeks * Represents newly-prepared sample. t Represents portion of sample used in preceding experiments and stored at F. for approximately months. Experiments and contained 0 New Hampshire male chicks per group. Experiment contained 0 cross-bred male chicks (N.H. c^xs.c. 9) per group. Growth data for experiments and were averaged. Downloaded from at Penn State University (Paterno Lib) on May, 0

5 SERUM CALCIUM AND ALBUMIN 87 nary studies using a phenol-water, twophase solubility system indicate the suitability of this system for countercurrent distribution procedures. Concentrates of the factor active at 0.00 to 0.0 percent of the diet were obtained. L-lyxoflavin failed to exhibit a growth response. A supplementary mixture of essential amino acids simulating those present in fish solubles also failed to elicit a growth response. These results indicate that the factor is distinct from L-lyxoflavin or the amino acids. REFERENCES Briggs, G. M., 9. A review of recent developments in poultry nutrition: Vitamin B, antibiotics, and new growth factors. Transactions American Association of Cereal Chemists, 0: -0. Bruins, H. W., M. L. Sunde, W. W. Cravens and E. E. Snell, 9. Growth-promoting activity of L-lyxoflavin. Proc. Soc. Exp. Biol. Med. 78: -. Combs, G. F., 9. Unidentified factors required for chick growth. Ninth World's Poultry Congress, : -9. Lillie, R. J., J. R. Sizemore and H. R. Bird, 9. Unidentified factors in poultry nutrition.. Development of a chick assay. Poultry Sci. : 8-8. Menge, H., G. F. Combs, P. T. Hsu and M. S. Shorb, 9a. Unidentified growth factors required by chicks and poults. I. Studies with chicks using purified diets. Poultry Sci. : 7-7. Menge, H., and G. F. Combs, 9b. Unidentified growth factors required by chicks and poults. II. Fractionation of a factor in liver. Poultry Sci. : Lack of a Correlation Between Variations in the Amount of Calcium and Serum Albumin in the Blood Sera of Chicks Contribution 89, Department of Chemistry. ROBERT E. CLEGG AND R. E. HEIN Kansas State College, Manhattan, Kansas (Received for publication February 0, 9) THE presence of more non-diffusible calcium in the sera of laying hens than in the sera of non-laying hens has been confirmed many times since it was reported by Correll and Hughes (9), and in more recent years others have shown that the same relationship holds for young hens and cockerels under the influence of the female sex hormone or diethylstilbestrol. In addition, Moore (98), Brandt et al. (9) and Clegg et al. (9) have demonstrated that the serum proteins of laying hens or hormone treated birds contain electrophoretic components not present in the serums of the normal non-laying birds. Some investigators, including Greenberg (9), are of the opinion that the serum albumin is the protein component responsible for most of the ion binding capacity of the blood serum. In the subsequent discussion, information concerning calcium binding ability of the serum components of chicken is discussed. EXPERIMENTAL The lack of a direct relationship bebetween the calcium binding capacity of chicken serum and the albumin concentration has been demonstrated in two ways. In the first case birds were fed a normal growing ration and, to days after diethylstilbestrol injections were started, the blood was collected. The sera Downloaded from at Penn State University (Paterno Lib) on May, 0