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Iodine reducing substances in thymus gland extracts Smith, David Burrard 1941

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IODINE REDUCING SUBSTANCES IN THYMUS GLAND EXTRACTS by David Burrard Smith A Thesis Submitted i n Partial Fulfilment of the Requirements for the Degree of MASTER OF ARTS i n the Department of CHEMISTRY The University of Bri t i s h Columbia April 1941 Acknowledgements I hereby wish to express my thanks to Dr. J. Allardycej at whose suggestion this research was initiated and under whose super-vision i t has been carried out, for much encouragement and advice. -I am also grateful to Drs. G. McQlean Frasera .and W. A. Clemens for allowing me laboratory space in the Department of Zoology. Acknowledgements are due to the Head of my own Department Dr. R. H. Clark for help during the past few years. I am deeply indebted to Mr. A. ¥. Lantz of Burns and Co. for much aid and interest. He was ready and vailing at a l l times to procure fresh glands and to see that they were properly kept until I. was able to fetch them. The work would hardly have been possible without his co-operation. Thanks are due to Burns and Co. for supplying the thymi used i n this work and also to the Vancouver Medical Association for permission to consult the literature i n their library. - i i -Table of Contents Introduction A. Embryology of the thymus B. Anatomical relations of the thymus C. Histology of the thymus D. Physiology of the thymus (a) Introduction (b) Involution (c) C l i n i c a l observations •(d) Effect of thymectomy (e) Connections with calcification (f) Connections with the gonads (g) Connections with the adrenals (h) Other endocrine relationships (i) Relationship with lymphatic system (j) As a source of nucleic acid (k) -Effect of feeding and injecting thymus substance (l) The work of Rowntree and his associates. E. Thymus preparations and extracts F. Effect of some sulfhydryl compounds and other reducing sub-stances on growth and development. G. Other reports of glutathione and ascorbic acid i n the thymus. Purpose of this research A. Plan of investigation - H i -fi. Review of methods of analysis (a) Glutathione (b) Cysteine (o) Ascorbic acid 3. Experimental work A. Preparation of extracts B. Determination of total I.R.T. G. Analysis for glutathione D. Analysis for cysteine E. Analysis for ascorbic acid F. Analysis of the extract 6. Determination of nitrogen distribution 4-. Discussion 5. Conclusion IODINE REDUCING SUBSTANCES IK THYMUS GLAND EXTRACT. In t r o d u c t i o n The thymus was given i t s name by Galen who named i t from the Greek 9V/xoS since i n the sheep i t seemed to resemble a bunch of thyme. I t I t i s s t i l l the enigmatic organ. I t remains the l a s t major mass of t i s s u e whose f u n c t i o n continues to defy e l u c i d a t i o n though i t gives h i n t s of p l a y i n g an important part i n the metabolism of the organism. Though i t i s a lymphoid s t r u c t u r e , i t d i f f e r s from other lymph glands both i n i t s embryonic o r i g i n and i n i t s h i s t o l o g i c s t r u c t u r e . I n the modern enthusiasm f o r endocrinology, many have sought t o in c l u d e i t among the glands of i n t e r n a l s e c r e t i o n . As s h a l l be seen, however, most questions regarding i t are only vaguely answered i f at a l l and have given r i s e to considerable controversy and contention. 'Embryology. The thymus bodies arise i n a l l vertebrates as one of the several sets of outgrowths from the pharyngial pouches. The number of pairs of thymic outgrowths varies widely both between and i n the different groups. In fishes, amphibians, reptiles and birds thymic tissue may arise from the second, third, fourth and f i f t h pouches (40, 52, 136, 143). The mammalian thymus i s derived from the third and fourth pouches. In most species, thymus IV i s only a rudimentary and transitory structure, but i n some,including the cat, i t always develops ( 7, 53, 143, 293). In a l l vertebrates the greater portion of the gland arises from entodermal epithelium of the medial and ventral portions of the third pouch. Its origin i s thus very similar to that of a number of other organs including the palatine tonsils from the second pouch, parathyroids from the dorsal walls of the third and fourth pouch and the ultimobranchial or lateral thyroid bodies from'*the f i f t h . The inferior pair of parathyroids ( the parathymus glands ) i n particular are closely associated with the upper end of the developing thymus. Parathyroid tissue i s frequently found embedded i n i t s upper parts. The mammalian thymus III anlages f i r s t show at the ten somites stage. At f i r s t hollow, they soon become solid epithelial strands The upper ends soon atrophy and undergo adsorption. Early i n uterine l i f e the cells lose their epithelial character and send out processes which anastomose to form a reticulum. At about this stage, the small thymic cells (see below) begin to appear and the gland slowly takes on the lymphoid appearance of later l i f e . Anatomical Relations Thymus bodies are found i n a l l vertebrates from cyclostomes to birds and mammals. In fishes i t remains i n a primitive condition i n that i t retains i t s connection with the epithelium from which i t arises. In the angler fish, i t forms a f l a t ovate placoid of epithelial and lymphoid tissue on the wall of the g i l l chamber (40). In the trout there are three thymic buds above the f i r s t , second and third g i l l arches (52). In tadpoles according to Speidal (256), the thymus consists of lymphoid ti-ssue interspersed with large blood sinuses. There i s no definite arrangement into cortex and medulla. In the frog. ( Rana pipens), the thymic bodies are a pair of compact masses on the sides of the head just posterior to the tympanic membrane. They show a ooctex and medulla. There i s no connection with the epithelium, the branchial pouches having disappeared. The glands are said to decrease i n size during metamorpnosis (221). / The glands vary i n shape i n the dil'ierent classes of reptiles. In snakes, the glands are elongated and extend along the carotids. In lizards and crocodiles, they are broad'and f l a t and extend along the carotids from the pericardium (190). In birds, the thymus proper consists of two rows of nodules lying on either side of the trachea. The number of nodules i s variable even i n the same species (50). Riddle (214, 217) believed that the bursa F a b r i c i i , a glandular pouch opening into the cloaca, i s also composed of thymic tissue and Greenwood (91) found similar tissue elsewhere in the body. The f u l l y developed human thymus i s roughly triangular i n -4-shape with i t s base resting on the pericardium. It extends from the level of the fourth costal cartilage to the lower t i p of the thyroid. Anteriorly i t i s i n contact, with the sternum and posteriorly, the trachea. It i s composed of two es each of which i s divided into lobules, 1-2 mm. i n diameter, giving the gland a warty appearance (90, 203). In .other mammals,-•the gland i s similar to that of man i n most respects. It varies i n the number of lobes, the size of the lobules, the relative amounts in the thorax and i n the neck and i n the frequency with which detached portions of thymic tissue are found (194). Histology On microscopic examination, the thymus i s found to consist of a cortex and a medulla. The cortex i s made up of densely packed masses of small cells morphologically identical with the small lymphocytes, supported by a network of reticulo-epithelial cells continuous with a similar network in J the medulla. The medulla i s marked off from the cortex by the abrupt disappearance of most of the small thymocytes. It also contains the very characteristic! structures, the concentric corpuscles of Hassail. The medulla i s more vascular than the cortex. The origin of the reticular cells from the original epith-e l i a l tissue can be traced embryologically. Their epithelial nature becomes prominent i n transplants and tissue cultures of the gland. The origin of the small thymocytes on the other hand i s not so settled. Most investigators regard them as lymphocytes which have invaded the epithelium since they resemble lymphocytes elsewhere i n a l l their morphological characteristics, i n their serological reactions, i n their susceptibility to roentgen ray injury and i n their general behaviour. -5-There are however some workers who regard the small thymic cells as derived with a l l other components of the gland from the original epithelial cells (92, 171,' 175, 176). Hassall's corpuscles are made up of large pearshaped cells arranged concentrically around a central core containing cellular debris, cysts and sometimes calcified material. They are probably spent and degenerating epithelial cells which have grown under unique conditions involving the loss of a surface from which desquamation could take place normally. Most investigators hold this view (53, 148, 171, 176, 293) though other interpretations are not lacking. Jordan et a l (137, 138) believe them to be due to occlusion of the lumen of medullary capillaries with a resulting lack of nourishment for the surrounding c e l l s . Occasionally i n the thymus of mammals there are found duct-like spaces lined with columnar or cuboidal epithelium. These when traced are found to end blindly. It has been suggested that they are due to remnants of the branchial pouch epithelium or due to failure of the origin-al thymus tubule to lose i t s lumen completely. There are also present i n the gland very variable numbers of mononuclear and polynuclear eosinophiles. The above remarks refer chiefly to the thymus of higher vertebrates. In the f i s h , the lymphoid character i s not so dominant and there are no Hassall's corpuscles (52). Physiology The function of the thymus i s s t i l l unknown. Data obtained from experiments involving thymus extirpation, implantation, feeding, injection of extracts as well as from c l i n i c a l observations have been interpreted differently by different observers, with the result that the physiology of the gland i s most vague. However, the facts that i t under-goes an involution beginning about puberty after increasing i n weight up to that time; that i t undergoes acute involution in starvation, pregnancy , severe disease or other adverse conditions; that after gonadeetomy i t s normal involution i s delayed; and that under other conditions such as Grave's disease, acromegaly and after adrenalectomy i t regenerates - a l l indicate that i t plays some important part i n metabolism at least up to the period of sexual maturity, even though i t i s not essential to l i f e . Involution One clear property of the thymus i s that i t i s relatively large i n infancy but that during maturity i t slowly regresses to practical-ly disappear i n old age. That i t reaches i t s greatest absolute weight at about the age of puberty i s certain. A general average of weights report-ed for the human thymus was given by Loewenberg (157) as follows: at birth 12 grams; at puberty (12-15 years) 35 grams; at 25 years, 23 grams; at 60 years 12 grams; at 70 years 6 grams. Others (33, 35, 293, 300) reported similar figures. Relative to the weight of the body, the thymus weight i s greatest at birth. Al a l l ages there is,moreover, a high individ-ual variation. After puberty, the gland atrophies, rapidly at f i r s t , and then more gradually, so that thymic tissue may be found even i n the most aged. This natural atrophy i s referred to as "age involution". The same essential picture of growth up to about puberty and atrophy there-after was the result of more complete and better controlled investigations on various animals, (Paton and Goodal, (199) on guinea pigs, Hatai (118) and Chiodi (45) on rats and others). Hammett (100, 102) however stated that involution does not begin until early adulthood but Haramar (99) pointed out that this increase after puberty of the whole gland did not exclude redaction of parenchyma. In the pigeo.K.i, Riddle and Fry (216) found that thymic involution began two months before sexual maturity measuring the latter by the beginning of egg laying fat about six months of age). It is also apparent that i n birds, i t degenerates more slowly than i n mammals (186). There i s much difference of opinion as to whether the gland i s at i t s maximum weight before, "at, or after the onset of puberty. More careful work w i l l have to be done to determine the exact moment, physiol-ogically, when involution begins (45, 100). The thymus i s a very labile organ. Under adverse body circumstances or when the organism i s subjected to unusual stress, i t undergoes an "accidental" involution, as opposed to the normal age involution. Malnutrition, starvation, operative shock, trauma, infections wasting diseases and pregnancy w i l l a l l cause accidental involution (157, 268). It decreases i n size i n most illnesses lasting more than twenty-four hours (33). According to Riddle and 5ry (216) the reduction of the thymus more speedily t e l l s of disease or adverse conditions i n Laboratory animals than the macroscopic examination of any other organ. Regeneration normally follows accidental involution. There are some histological differences between the two types of involution. In age involution there is a decrease i n the number of Hassal^s corpuscles, while i n accidental involution there i s an increase. C l i n i c a l Observations This aspect has been reviewed by Loewenberg ( 157) and numerous reports are to be found i n medical literature. The thymus may Be involved i n various abnormal conditions - i t may be enlarged or atrophied, i t may be the seat of inflammation, abscess or hemorrhage, i t may be infected by syphilis, tuberculosis, actinomycosis etc., or i t may be the seat of malignant or benign tumors - but there seems to be no disease which can be ascribed to disfunction of the gland i t s e l f . It i s affected i n many endocrinopathies but in a l l cases apparently only secondarily (92, 300). There have been, however, reports that partial ablation of overlarge thymi has corrected retarded mental and physical development (36), The term Mors thymica has been applied to instances of sudden death of infants who had previously been i n apparently good health and i n whom the only abnormal finding has been an enlarged thymus. The cause of such a tragedyhas usually been ascribed to either pressure of the gland on trachea or heart or to anaphylactic' phenomena. The most celebrated synd»me involving the thymus i s that known as Status thymicolymphaticus. The anomaly i s an indefinite one. One of the symptoms i s a hyperplasia of the lymphatic structures, including the thymus, but there are also manifest numerous other disturbances involving the gonads, the cardio-vascular system, the gastrointestinal tract etc.. The basal metabolic rate i s low, there i s a tendency to allergic phenomena and a greater susceptibility to infection. Most of the pathological relations of the thymus emphasize i t s similarities with other lymphoid tissues. It was early demonstrated that the thymus was not essential for l i f e . The i l l effects, including tetany and abnormal bone development obtained by some early investigators have been ascribed to infections, malnutrition or to unwitting removal of the parathyroids (194). By far the greater number of more recent experiments on various animals, including the rat (191), the guinea pig (199), the rabbit (169, 284), the dog (194), the pigeon or chicken (174, 186, 217) have showed that thymectomy was not followed by detectable symptoms i n otherwise normal animals. A few investigators have found changes i n metabolism after thymectomy. Sandberg, Perla and Holly (232) reported that urinary nitroge increases from 13^ ti£ intake to 40% twelve weeks after the operation, the increase being accounted for by urea. Riddle and Braucher (215) found an erythrocyte count 16/, higher than the normal in pigeons from which the thymus and bursa had been removed. Miller (183) reported calcium abnor-malities i n thymectomized hens but here the probability seems to be tnat the parathyroids were involved. In general, the conclusion has been widely drawn that the functions oi" the thymus may be performed or compensated for by other lymphoid tissues. It has been shown (89) that remnants oi" the thymus may hypertrophy after the removal of the major portion of the gland. Connections with calcification On the basis of a close embryonic origin, a relationship between the thymus and the parathyroids has been thought lik e l y . This -10» belief was reinforced by the disturbances in calcium metabolism which sometimes resulted from the extirpation experiments of some early workers -. More recently others have associated the thymus with calcification. Riddle (213) found tnat some pigeons whicn laid eggs without shells, had atrophied thymus. He believed that there was a hormone acting on the oviduct ana named i t Thymovidm. He could not reproduce the condition by removing the thymus, blaming this failure on rather hypothetical (see 91) thymic tissue elsewhere m the body. Others have also obtained similar negative results (1, 186;. Glaessner and Hass (82) stated that regeneration of fractured bones was delayed i n thymectomized cats but that i t may be stimulated by the injection of a thymus extract. Harris (114), finding that the thymus atrophied in hypervitam-inosis D concluded that i t had some role i n calcium metabolism. He also stated that thymus implants increased the density of the bones. He has made a short review of the evidence linking the thymus and calcification. Rowntree, Clark and Hanson (233) reported a rise i n blood calcium ai d phosphorus with accelerated bone development in the offspring of rats treated with Hanson's thymus extract (see below). On the other hand other investigators (193) found no such evidence. Park and HcClure (194) i n an extensive review and after careful experiments on dogs noted no changes whatever after thymectomy. They attributed previous results to parathyroidectomy, varying degrees of rickets and other disturbances not connected with the thymus. Thomson and Collip (277) state that they remain unconvinced that any definite role of the thymus i n calcification has been established. -11-Connection with the Gonads. The most obvious property of the thymus i s that of starting to regress apparently just at the age when sexual glands are beginning to function. This has led many workers to believe that there i s an intimate relationship between the thymus and the gonads ana to plan experiments accordingly. Many of the results of these have been reviewed by Andersen (5). A few investigators have reported changes i n the gonads following thymectomy. Paton (196, 197, 198), found heavier testes i n young operated rats than i n controls but no difference i n adults. Others have reported a decrease i n the size of the gonads but these results appear to have been due to the poor conditions of the animals. Most workers report no change whatever i n the sex organs after removal of the thymus. Hainan and Marshall (98) and Ronton (211) with guinea pigs, Pappenheimer (191), Reinhardt (210), Plagge (204), Segaloff and Nelson (241) and Anderson (6) with rats, Masui and Tamura (173) on mice, Park and McGlure (194) with dogs, Riddle and Krizenecky (217), Achert and Morris ( l ) , Morgan and Grierson (186) on birds and Allen (3) on tadpoles, a l l obtained negative results. They found no significant deviations from the normal in the factors noted, including size and weight of gonads, spermatogenesis or commencement of ovulations It has also been shown that thymectomy had noteffect on pregnancy (6, 199, 241). The conclusion i s that extirpation of the thymus has no eifect on the development and function of the reproductive organs. On tne other hand extirpation of the gonads has been shown to have a definite effect on the thymus.' The normal age involution was delayed and slowed. This delay was most marked after prepuberal castration but was also measurable i n adult castrates. One investigator, only, Paton (.197 J reported no difference following spaying i n guinea pigs. A l l others (7b, 88, 91, 98, 120, 170, 296) i n giving their observations on rats, guinea pigs, fowls, heifers agreed that the normal atrophy of tne thymus was delayed though not prevented by gonadectomy. Gmodi (4aJ determined the growth curve of the thymus in normal and i n castrated rats. He showed that i n animals castrated before puberty this curve was parallel to, though higher, than the normal. He gave as his opinion that since the general shape of the thymus growth curve was not affected by castration that the involution of the thymus was not influenced by the development of the sex glands. Results obtained by Evans and Simpson (66) showed that the effect of castration was due to the removal of the i n t e r s t i t i a l cells, since destruction of the germinal epithelium by operative cryptorchidism or by vitamin E deficiency caused no abnormalities i n thymus development. Biddelph and Meyer (20) however, reported a delay of thymus involution inthe male, but not female, rat on a vitamin E deficient diet. The, delay was not great. Hammar (99) stated that the injection of the serum of mature animals into immature ones of the same species caused thymus involution. This effect was not found on injecting the serum of immature animals. Moreover, atrophy of the thymus was produced by the inject-ion oi sex hormones and of gonadotropic substances into both prepuberal and mature animals. Also the effect of gonadectomy could be overcome by the injection of sex hox-mones i n suitable amounts. Ovarian, placental and testicular normones a l l caused apparently the same effect i n animals regardless of sex. Allen i n 1928 (4) found that the thymus of monkeys injected with an ovarian extract weighed less than those of controls. Golding and Ramirez (87) injected aqueous extracts of ovary and placenta into rats and obtained a similar result. Extracts of mare's uterus and placenta were also potent (66) as was a testicular hormone obtained from urine (150, 151). That the effect was actually due to the sex hormone was proved by the use of more or less purified preparations and also of synthetic hormones and substances oi' closely related structure. Estrone or theelin, estrin, estradiol, testosterone propionate and the "unnatural" ahdrostenedione and androstenediol have caused atrophy of the thymus in normal and gonadectonized rats of both sexes. If administered i n suitable amounts some of these hormones have prevented castration hypertrophy of the thymus (45, 7 6 , 150, 151, 152, 154, 202, .-296). Only Low (159) reported negative results on injection of theelin. Butcher and Persike (42) used a pituitary gonadotropic preparation Antuitrin S. This caused thymic regression i n normal rats over 17-18 days of age. It had no effect oh gonadectomized animals. These results are very definite and seem to show conclusiv-ely that the increase i n the sex hormone content of the circulation (corresponding to the onset of puberty ) is an important factor i n the normal age involution of the thymus. Many cases have been reported of thymus hyperplasia assoc-iated with hypogonadism, cryptorchidism and similar conditions (157) and much has been made of them as pointing towards the function of the thymus. -14-But i n the light of recent research, i t would appear that the thymus was only secondarily involved. It' can therefore be saia that, to date, no definite relation-ship between the gonads and the thymus has been demonstrated. Connection of the Thymus with the Adrenals Another reported interrelation of the thymus has been with the adrenals. Wiessel (294) i n 1905 noted that i n status lymphaticus, there are frequently lesions of the adrenals, also the atrophy of the adrenals i n Addison's disease i s associated with a persistant thymus. Other c l i n i c a l observations (132„ 191, 240) have been along the same lines. Experimental work has also indicated some sort of mutual antagonism between the two glands. In 1914 Crowe and Wislocki (49) obtained a generalized hyperplasia of lymphoid tissue following the removal of the adrenal cortex. Jaffe (134) and Marine, Manley and Baumann (l'/u) obtained the same effect. Richter and Wislocki (212) while investigating the changes produced by hypophysectomy found that while the adrenals were hypoplastic,the thymus and lymph glands of The whole body were markedly enlarged. According to Chiodi (45), i n rats which had been castrated prepuberally and adrenalectomized at 80-100 days of age, there was only a slight increase i n thymus weight as compared with control castrates. Selye (242) reported the converse results that enlarged adrenals caused an atrophy i n the thymus. The injection of cortin would bring about this atrophy with or without the presence of adrenals. Ingle (133) gave rats cortin i n their drinking water and obtained an involution oi the thymus. Low (59) however reported negative results from the injection of an adrenal cortical preparation called Eschatm. -15-Th© administration oi' pituitary adrenocorticotropic hormone was said to cause thymus regression i i " the adrenals were present (67, 270). Investigation on this apparent relationship by working i'rom tne tnymus enu, have not been so productive. Here also the removal of the"thymus has given l i t t l e hint as to i t s function. Park and McClure ,^194) i n their very thorough researcn got no change i n the adrenals after thymectomy. Segaloi'f ana Nelson (240) thought that thymectomy might have an effect on the course of adrenal insufficiency in the rat. They therefore injected cortm into adrenalectomized rats irom some of which the tnymus was removed also. There was no duxerence m the growtn cux-ves o£ the two groups. On the other hand Rowntree (222) wrote that the administrat-ion of Hanson!s thymus extract (see below) led to hypoplasia of the rat adrenal. Gershon-Cohen et a l (80) destroyed much of the thymus by X-ray irradiation and report adrenal enlargement, but this treatment was perhaps a violent one.. From the above i t i s seen that experiments on this aspect of thymwsfunction, while making i t certain that adrenal impairment i s associated with hypertrophy or at least regeneration of the thymus, have not shown definitely just what this connection i s . Connection of the. Thymus with the Thyroid Based on the c l i n i c a l observation that i n exophthalmic goiter there i s often enlargement of the thymus (181, 168, 290), a relation has been sought with the thyroid. Speidal (256) has compared a hyperplasia of lymphocytes i n the thymi of tadpoles which had been administered. -16-thyroid substance, with the thymus enlargement mentioned above. It has also bean shown that thyroidectomy hastens the involution of the thymus and also prevents the regeneration normally following adrenalectomy (l'/0) In Hammett's view, however, these effects may well be due to the general physiological disturbance and not to any specific relationship (100,101). He stated that the main factor i n the growth of the thymus i s i t s close connection with the general bodily condition. In the opinion of Williamson and Fearce (295), the thymus i s essentially a lymph reservoir for the thyroid. GhouKe,Whitehead and Parker (46) ,• however, could find, no trace of any such lymphatic system i n human cadavers or i n dogs. Nor could Weller find any embryologies! evidence of i t (293). Connections with other Endocrine glands No definite interrelationships with the other endocrines have been made out. &fter hypophysectomy i t has been reported that the thymus enlarged (212) but i t has also been reported to atrophy (253,67). Pituitary gonadotropic hormone caused thymus involution but presumably through the medium of the sex glands. Low (159) obtained no effect follow-ing the: injection of an anterior pituitary extract (alpha and beta c e l l hormones). In this regard, it*might be mentioned that Low injected several endocrine, extracts into rats and got negative effects in a l l cases (see above). He admits that his extracts were only "supposed" to contain hormones, however. Some of them were commercial preparations. B'omshov and Sladovic (32) have made the most recent claim to have demonstrated a thymus hormone. It was obtained from the lipoidal -17-i' r a c t i o n . Xts a d m i n i s t r a t i o n was stated to cause disappearance of glycogen from the l i v e r of r a t s . They ascribed the s t i m u l a t i n g a c t i o n of the thymus on growth to the m o b i l i z a t i o n of t h i s carbohydrate. This antogonism to the I s l e t s of Langerhans has been reported e a r l i e r by one or two others, i but has not been investigs&ed thoroughly (157). Summary of Endocrine Connections From the above, i t i s seen that no d e f i n i t e and s p e c i f i c i n t e r r e l a t i o n s h i p between the thymus and and any of the glands of i n t e r n a l "^Secretion has been e s t a b l i s h e d . Though the above review i s f a r from being complete, so voluminous i s the l i t e r a t u r e on the thymus, even i f i t were more comprehensive the conclusion j u s t stated would be unchanged. I t has not been proved that the thymus i t s e l f has any i n t e r n a l s e c r e t i o n . There are some a u t h o r i t i e s who deny that i t has any endocrine p r o p e r t i e s whatever. -H contains no groups of c e l l s which seem l i k e l y to be s e c r e t o r y . The greater number of i t s c e l l s are thymocytes which are c l o s e l y r e l a t e d t o , i f not i d e n t i c a l w i t h , lymphocytes. The purpose of the r e t i c u l a r c e l l s i s , apparently, to provide a support f o r the thymocytes. The c e l l s of H a s s a l l ' s corpuscles must be looked upon as degenerating r e t i c u l a r c e l l s . They are h y a l i n i z e d and may undergo l i j t t l f a c t i o n or become c y s t i c or c a l c i f i e d . The c y s t i c ducts are found only very o c c a s i o n a l l y (The greatest frequency i s i n dog's thymi where i t i s 20%). Rslationshi.T^-Jia-kainjhoid t i s s u s s -Though the thymus i s of e p i t h e l i a l o r i g i n i t i s changed very e a r l y i n p r e n a t a l l i f e so as to become lymphoid i n appearance. I t i s also a f a c t t h a t in.most of I t s p h y s i o l o g i c a l and p a t h o l o g i c a l r e a c t i o n s i t resembles or d i n a r y lymphoid t i s s u e (175). -18-On the nature oi' the thymocytes hlaximow (176) w r i t e s as f o l l o w s "They are morphologically i d e n t i c a l with lymphocytes. Both show the same s u s c e p t i b i l i t y to X-ray i n j u r y ; both are c'ytolyzed by sera obtained by the i n j e c t i o n of thymus c e l l s i n t o r a t s (192); and both show the same type of ameboid motion. Gregoire has shown that t r a n s p l a n t s of the thymus c o n s i s t only of epithelium If lymphocytes are prevented from migrating i n t o them by mechanical means. Further, the transformation of the small thymocytes i n t o plasma c e l l s and e o s i n o p h i l myelocytes i s g e n e r a l l y admitted. The mitochondria have the same appearance i n both types of c e l l s . However i n s p i t e of these s i m i l a r i t i e s , a few authors are u n w i l l i n g to c l a s s i f y the small thymocytes as lymphocytes because they b e l i e v e the thymocytes to have an e p i t h e l i a l o r i g i n , although most workers b e l i e v e t h a t they a r i s e from lymphocytes which have wandered i n t o the epithelium" (176). I n ordinary lymph t i s s u e s an age i n v o l u t i o n s i m i l a r to t h a t of the thymus begins about the age of puberty, lymphatic t i s s u e being normally prominent i n c h i l d r e n . Regeneration of lymph glands occurs a f t e r adrenalectomy (134). Ghiodi (45) found that the weights of the submaxillary, c e r v i c a l and a x i l l a r y lymphatic g a n g l i a were s l i g h t l y higher i n c a s t r a t e s as compared with c o n t r o l s , p a r a l l e l i n g the e f f e c t of the same operation on the thymus. I n Grave's disease and i n acromegaly where -..a there i s thymus enlargement there I s a s i m i l a r enlargement of other lymphoid organs, such as lymph glands, t o n s i l s and spleen. I n the c o n d i t i o n known as Status lymphatic us, •- , though the most prominent symptom i s the enlarged thymus, there i s hypertrophy of a l l lymphoid and lymphatic t i s s u e s i n the body. These and other resemblances and the f a c t that the organism can apparently w e l l get along without i t s thymus have l e d to tte. view that the thymus i s p r i m a r i l y part of the lymphatic system. This does not solve the problem of i t s f u n c t i o n since the f u n c t i o n of lymphoid t i s s u e s i s s t i l l i n d e f i n i t e l y known but i t opens up other avenues of approach to the thymus problem. The fa.notions of lymphoid t i s s u e s are g e n e r a l l y s a i d to be the production of lymphocytes, f i l t r a t i o n and a c e r t a i n amount of p u r i f i c a t i o n of the blood and some clos e a s s o c i a t i o n w i t h the defense r e a c t i o n s of the organism such as the e l a b o r a t i o n of a n t i b o d i e s . F r i e d l a n d e r (74) stated that the thymus produces lymphocytes. Maximow (175) and Marine (171) hold s i m i l a r opinions. There has been considerable work done on the r o l e lymphoid t i s s u e p l a y s i n r e s i s t a n c e to i n f e c t i o n s and cancer and some experiment-ers have i n v e s t i g a t e d the thymus along t h i s l i n e . A f t e r adrenalectomy when there has been hypertrophy of the thymus, i t has been observed that antibodies were formed more r a p i d l y than i n normal animals although these were more s u s c e p t i b l e t o t o x i n s (273). Loeweriherg (157) expressed the opinion t h a t the thymus operated to prevent pulmonary t u b e r c u l o s i s i n prepuberal c h i l d r e n . He pointed out th a t t h i s disease i s rare or mil d i n cn i l d h o o d . Pearce and van A l l e n (200) i n v e s t i g a t e d the e f f e c t of thymectomy on s y p h i l i s i n the r a b b i t . They found, however, that the disease was .milder than i n normal c o n t r o l s . Many cases of sudden death have followed the i n j e c t i o n of u s u a l l y safe amounts of antiserum. Antopsy has shown the cause of death to be Status lymphaticus. Persons s u f f e r i n g from t h i s disorder have a tendency towards asthma, a l l e r g i c a l r e a c t i o n s and other p r o t e i n -20-s e n s i t l v i t y (157). With these s i m i l a r i t i e s there remains the d i f f e r e n t o r i g i n of the thymus from other lymph organs (the p a l a t i n e t o n s i l s would appear to • havena somewhat s i m i l a r or.i.gin to the thymus) . There i s also the anatomical d i f f e r e n c e i n the absence of the t y p i c a l lymph nodules and lymph sinuses possessed by other lymphoid organs (237), 3y v i r t u e of the close s i m i l a r i t i e s of the thymus with other lymphatic organs, I t I s very reasonable to assume that I t Is p r i m a r i l y " part of that system. But i t Is s t i l l not unreasonable, on account of the d i f f e r e n c e s , to expect the gland t o d i s p l a y p r o p e r t i e s d i f f e r e n t i n kina or degree from the other components of the system. The most recent researches however, have f a i l e d to i n d i c a t e d e f i n i t e l y what these prop-e r t i e s might be.' Role as regards K u c l e i c A c i d The f a c t t h a t the thymus contains a greater concentration of * thymonucleic a c i d than other t i s s u e s has l e d t o the conjecture that I t acts as a n u c l e i n r e g u l a t o r or as a store of n u c l e i c a c i d (I35a). I t would appear, though, t h a t the densely packed thymocytes with t h e i r email amount of cytoplasm and r e l a t i v e l y l a r ge n u c l e i might account f o r the l a r g e r amount of n u c l e i c a c i d . Ef f ecjL-aiL Feeding or I n j e c t i n g Thymic Substance on Growth and Development. Among the f i r s t attempts to e l u c i d a t e thymus f u n c t i o n by feeding the gland to various animals yere those of Gudernatsch with tadpoles. The t r e a t e d tadpoles, he reported, grew very l a r g e and d i d not metamorphose (95). Ulleahuth (281,282,233) got developmental disturbances -21-w i t h tadpoles which were fed on an e x c l u s i v e l y thymus d i e t . He l a t e r showed that these were due to the inadequacy of the d i e t since tadpoles f e d supplementary plant food developed normally. Some of the expe* iments on the thymus feeding of animals also f a i l e d f o r the same reason. Hewer (125) i n 1916 fed r a t s on thymus and reported an absence of spermatogenesis. In the l i g h t of present knowledge t h i s experiment showed that the gland was l a c k i n g i n Vitamin E. Hoskins t r i e d the same experiment, i n c l u d i n g other food, s t u f f s i n the d i e t and got r a t s which were apparently quite normal (129, 130). Gudernatch (96) however, reported t h a t thymus fed r a t s grew more r a p i d l y and were more vigorous than h i s c o n t r o l s . On the other hand, MacKay and Barnes (165) found t h a t thymus feeding depressed the growth curve considerably. As mentioned e a r l i e r R i d d l e (213) corrected the l a y i n g of soft s h e l l e d eggs by pigeons by feeding bovine thymus. Others (1,186) have been unable to confirm these r e s u l t s . On the whole, t h e r e f o r e , the evidence I s against the thymus, having any s p e c i f i c e f f e c t as a food. E x t r a c t s of the Thymus Gland Various e x t r a c t s have been prepared from the thymus but the a d m i n i s t r a t i o n of these to la b o r a t o r y animals has given widely varying r e s u l t s I n d i f f e r e n t hands. The f i r s t of these e x t r a c t s was prepared by Asher, who c a l l e d i t Thymocresin (157) This e x t r a c t when i n j e c t e d Intramuscularly d a i l y i n one m i l l i g r a m doses Increased the rate of growth of r a t s and also kept them a l i v e on a vi t a m i n - f r e e d i e t on which the c o n t r o l s died ( 8 ) . Segaloff and Nelson (239), however, prepared an ex t r a c t according to -22-Asher's d i r e c t i o n s , having the same q u a l i t a t i v e t e s t s . They i n j e c t e d f i v e times the minimum dose reported by Asher and got no r e s u l t s . They aontinued the treatment over s i x successive generations and could not f i n d any v a r i a t i o n s i n growth and development. Another e x t r a c t was introduced by Temesvary (276). % employed i t i n strengthening and prolonging the u t e r i n e c o n t r a c t i o n s i n the e a r l y stages of l a b o r . L a t e r t h i s e x t r a c t was improved by adding p i t u i t r i n from the. p o s t e r i o r lobe of the p i t u i t a r y . I t has, however, been found that other t i s s u e s could replace the thymus (135). This combination of thymus and p i t u i t a r y e x t r a c t s i s c a l l e d Thymophysin. Downs and Eddy (54) i n j e c t e d r a t s w i t h a s a l i n e suspension of desiccated thymus and could f i n d no d i f f e r e n c e from s a l i n e i n j e c t e d c o n t r o l s . I n 1930 Hanson prepared an extract which he used i n the a l l e v i a t i o n of some cowers (110) . The hypothesis behind t h i s i s that c a i c e r i s caused -by an unbalance between growth promoting elements and " a n t i p l a s t i c agencies. E x t r a c t s of thymus, spleen and the "organs aretarsed to increase these a l l e g e d a n t i p l a s t i c agencies. Hanson reported that t h i s e x t r a c t , which he c a l l e d K a r k i n o l y s i n , had been used with good e f f e c t s on some cancer cases ( H Q , H I ) . The Work of Rowntree and h i s C o l l a b o r a t o r s We now come to consider the work of Dr. llowntree and h i s c o l l a b o r a t o r s at the P h i l a d e l p h i a I n s t i t u t e f o r Medical Research. Hanson o r i g i n a l l y supplied Rowntree w i t h a quantity of h i s e x t r a c t , the i n t e n t i o n being that the I n s t i t u t e would sake t e s t s on f r e s h batches as they were made. The procedure that Rowntree and h i s co-workers, Clark and -23-Steinberg, adopted was to I n j e c t I cc. d a i l y of the e x t r a c t i n t r a p a r i t -o n e a l l y i n t o successive generations of r a t s . The r e s u l t s they reported -.were; amazing. These r e s u l t s were summed up i n the f o l l o w i n g quotation "Following'the continuous a d m i n i s t r a t i o n of thymus e x t r a c t to successive, generations of parents, marked a c c e l e r a t i o n i n the rate of growth and development has been observed during the e a r l y l i f e of the o f f s p r i n g , p a r t i c u l a r l y of the t h i r d and l a t e r generations. Thus the rate of development encountered i n the f i f t h generation of young rsfc s born of four generations of thymus t r e a t e d forbears i s almost beyond b e l i e f " (226). Also see.(222,223,224,225). This accelerated development I s shown i n the f o l l o w i n g t a b l e from (226)*-Controls F4 Average b i r t h weight (grams) 4.6 5,1" 5.3 5.3 5.6 Ear opened (days) 2vr-3|r 2 1-2 I I Teeth erupted (days) 8-10 1-9 1-2 I I H a i r appeared (days) 12-16 3-12 4-6 4-5 . .2-3. Eyes opened (days) 14-17 12-14 4-6 4-6 2-3 Testes descended (days) 35-40 15-29 5-21 5-12 4-5 Vagina opened (days) 55-62 30-45 23-32 21-27 18-19 The low numbers u s u a l l y r e l a t e to the l a t e l i t t e r s i n the generation and the high numbers to the f i r s t l i t t e r s born. The growth curves showed an accruing a c c e l e r a t i o n i n weight i n each succeeding generation. S'or example the f i g u r e s f o r the F^ generation at 25 days of age were 37 grams f o r the c o n t r o l s and 94 grams f o r the -24- . . . . . . . . tests. Giant rats did not result however, the waight curves of tests and controls approaching after the 60th day. Eventually in f u l l y mature rats l i t t l e difference was apparent. With the acceleration of physical development there went a psychic precocity also. The F^ rats could look after themselves as well at 3 days of age as normal rats of 16 to 20 days. Weaning was possible at 3 days of age. The effect of the extract was largely confined to the growth and development of the young but Rowntree also reported a somewhat shorter period between l i t t e r s i n thymus treated parents. The result of omitting the injections for one generation i n a Beries was the entire disappearance of the precocity built up i n previous generations. They also reported that large doses of the extract were toxic, causing death through complete auricular-ventricular heart block. X-ray studies on the developing skeleton disclosed that the bones lengthened more rapidly than normally and ossification set i n much earlier. This was correlated with an increase i n the calcium and phosphorus content of the blood. The control rats had a calcium content of 9 to 11 mg. % while i n the tests the serum calcium increased to 13.3 mg in the F4 generation. The corresponging figures for phosphorus were 3-4 mg. % and 7.5 mg. % (222). Small adrenals, lymphatic hyperplasia and eosinophilic hyperplasia of the pituitary were other effects reported. Also Buckley reported accelerated development in the nervous -25-systems oi' treated rats (38, 39), Normally only scattered myelinated fibers are found at the end of 48 hours after birth i n the rat, Buckley, however, reported that he found myeliniaation i n the spinal cords of 6 day old rats which were descended from thymus treated forbears, equivalent to that of 13 Gay old normal animals. He later repeated this invest-igation using a new born rat of Rowntree's F ^ thymus treated generation. There was myelinization far i n advance of controls of the same age. Although i t was reported (227) that the injection experiments were continued through twelve generations with cumulative precocity i n each generation, no figures have been published on the development of the last l i t t e r which must have been approaching a limit. A decrease i n the average period between casting of l i t t e r s was reported for the tests. It was irregular, though, and apparently not accruing. No data was given relative to a shortening of the period of gestation which might have been expected. Einhorn and Rowntree (60) have observed the effect of removal of the thymus at an early age from successive generations of rats. They have noted retardation of development though i t was not as striking as the acceleration on administering extract. The effect of implanting thymi into rats has also been tried (59, 61). Homologous implants were made weekly, twelve being made m each rat. This was continued for four generations. Precocity was not so marked as that following daily injections of 1 cc of Hanson's extract. Som -26-data i s i n the f o l l o w i n g t a b l e . Bars 5eeth Eyes Tested Vagina opened erupted opened descended opened Controls 2.9 8.5 15.2 30.2 44.3 E 4 generation 2 5 7 24.3 36 Time i n days. They also observed the e f f e c t of thymus implanting (58) and the i n j e c t i n g of e x t r a c t (57) on thymectomized r a t s . In both cases the retarding' e f f e c t of thymectomy was overcome and was changed to an accruing a c c e l e r a t i o n , f r e q u e n t homologous implants were found to be more e f f e c t i v e than the a d m i n i s t r a t i o n of c a l f thymus e x t r a c t . These very d i s t i n c t i v e and most remarkable f i n d i n g s required c o n f i r m a t i o n . Other i n v e s t i g a t o r s however, have been unable t o reproduce them. C h i o d i i n 1938 (44) and Segaloff and Nelson i n 1940 (241) have a l l t r i e d the e f f e c t of thymectomy at an e a r l y age on r a t s over f i v e or s i x successive generations. The l a s t named removed the thymus at the age of 21 days over s i x generations. The ra t e of growth of both males and females was unaltered, and there was no departure from the normal In any of the developmental events s t u d i e d . Putzu (205) reported that no a l t e r a t i o n i n the somatic or psychic development occured i n the progeny of f i v e consec-u t i v e generations of thymectomized r a b b i t s . I n j e c t i o n of e x t r a c t s of thymus also produced only negative r e s u l t s . C h i o d i (43) made up an e x t r a c t weekly according to Hanson's method and kept i t f r o z e n u n t i l i t was to be used. The weight curves up to 40 days of age d i d not vary from those of the c o n t r o l s i n the f i v e gener-ations he t r e a t e d ; neither was there any s i g n i f i c a n t d i f f e r e n c e i n -27-development. Smith and Jones (254) also could detect no v a r i a t i o n s fro© the normal i n ^ the growth of mice over 5 generations. There was, however a marked lowering of f e r t i l i t y i n the t r e a t e d o f f s p r i n g of t r e a t e d mice. I n p a r t i c u l a r , Miss. U r s u l a Dale of the Department of Zoology of t h i s U n i v e r s i t y i n an attempt to reproduce Rowntree*s r e s u l t s , could f i n d no evidence of any a c c e l e r a t i o n of growth or development at the end of f i v e generations. F u r t h e r , she was able to detect no d i f f e r e n c e i n s i z e , s t r u c t u r e or h i s t o l o g i c a l appearance i n the p i t u i t a r y , p i n e a l , t h y r o i d , parathyroids, thymus, pancreas, adrenals, t e s t e s or ovaries of the treated r a t s (51,2) . ' B u r r i l l and Ivy (41) worked on the theory that treatment during pregnancy might have speeded up the metabolic rates of the f e t u s and t h a t , a f t e r b i r t h , I t might have continued to obtain the s t i m u l a t i n g p r i n c i p l e through the milk of the mother (though Rowntree (225,226) stated that i t was not t r a n s f e r r e d I n t h i s way). This a l t e r e d metabolic rate might be r e t a i n e d and the fetuses of ths next generation would be subject to a double e f f e c t , Rowntree (230) had stated that treatment of the males was unnecessary. B u r r i l l and Ivy t h e r e f o r e t e s t e d to determine whether the reported e f f e c t s could be produced by daLly i n j e c t i o n s of ground thymus t i s s u e i n t o females during pregnancy only. The experiment was continued f o r four- generations. Normal l i t t e r s r e s u l t e d ana no s i g n i f i c a n t d i f f e r e n c e * over the c o n t r o l s were reported. The continued lack of success i n thymus experiments along Rowntree 5s l i n e , e s p e c i a l l y with regard to the e f f e c t s of thymectomy, where the d i f f i c u l t i e s connected with the l a b i l i t y of the e x t r a c t (see below) would not a r i s e , have cast considerable doubt on the v a l i d i t y of h i s f i n d -i n g s . -28-. Hanson, however, stated that he f e l t assured that the r e s u l t s were a l l that had been claimed ( I I I ) . Loewenberg reported that he had seen the o r i g i n a l r a t and a l s o the members of twelve successive t r e a t e d generations.(157). He was " t r u l y astonished" at the p r e c o c i t y of members of the t w e l f t h generation, ^t'^also recorded that Rowntree. Clark and Steinberg gave a motion p i c t u r e demonstration of t h e i r work to the 48th Annual meeting of the American P h y s i o l o g i c a l Society at "Washington D.C. i n March 1936 (228). Buckley (38) observed i n the process of b i r t h an F J I thymus, treated' r a t i n which he found myelinated f i b e r s on examin-i n g i t immediately t h e r e a f t e r . P r o p e r t i e s of the e x t r a c t s us.ed by Rowntree. The extract used by Rowntree i n h i s e a r l y experiments was one prepared by Hanson i n 1930, whose method was as follows (110). Fresh thymi. from the neck of calves between 2-6 weeks of age'were ground to a t h i c k paste. This was'added to 1% HC1 i n the * p r o p o r t i o n of 0.6 gr. gland to I cc HG1. The mixture was s t i r r e d and slowly heated so that i t took I-g- hours to reach a temperature of 94°G. I t was then cooled and f i l t e r e d . The f i l t r a t e was made up to 990cc by pouring hot d i s t i l l e d water over the r e s i d u e . 10 cc. of 3% menthol In 95)« e t h y l a l c o h o l was added as a p r e s e r v a t i v e and the extract was b o t t l e d i n glass v i a l s , 'fhe pH was about 5.). Now the f i r s t batch of e x t r act supplied to Rowntree remaned b i o l o g i c a l l y potent f o r four years. I t was, more over, the only batch of Hanson 5s e x t r a c t to be reported as anything but very unstable. Hanson himself wrote that he was at a l o s s to account f o r i t s s t a b i l i t y (112). -29-E a r l y i n 1934, a new preparation was received from Hanson. The r e s u l t s obtained w i t h t h i s were e n t i r e l y negative, no p r e c o c i t y being noted i n the t h i r d generation (225, 226). Later the P h i l a d e l p h i a workers prepared t h e i r own extract a f t e r Hanson's method. They obtained the glands from mil k - f e d calves .and made up the e x t r a c t immediately. This was done weekly. The f i r s t batch of extract contained o x i d i s e d and reduced s u l f u r compounds to the amount of I5mg. % c a l c u l a t e d as g l u t a t h i o n e . The second, impotent e x t r a c t contained p r a c t i c a l l y none. Hanson reported h i s f r e s h product to have an iodine reducing t i t r e of over 100 mg.{£ gl u t a t h i o n e . I t contained 2-4 c/a p r o t e i n . The f a c t that reducing substances, e s p e c i a l l y sulphydryl, were to be found I n a c t i v e e x t r a c t s but not i n i n a c t i v e , became an important f a c t o r i n d i r e c t i n g the course of Rowntree*s experiments (229). I t was b e l i e v e d that there was a p o s i t i v e c o r r e l a t i o n between a high i o d i n e reducing t i t r e ( i . r . t . ) and the e f f i c i e n c y of c o l l e c t i o n . With t h i s i n mind Steinberg, one of Rowntree's coworkers, so modified Hanson's method that the co n d i t i o n s were more favorable f o r g e t t i n g a high i . r . t . . Weinberg's method Involved heating f r e s h ground thymus glands with T/a HC1 r a p i d l y w i t h s t i r r i n g to 68 C. I t was f i l t e r e d and b o t t l e d . 0.2/i chl o r b u t a n o l was used as the p r e s e r v a t i v e . The pr o p o r t i o n of gland to a c i d was 1.5 gr. to 1.0 cc.. This extract had an i . r . t . of between 200-400 m.g.% g l u t a t h i o n e . I t had a n e g l i g i b l e p r o t e i n content (258). *m a n a l y s i s of Steinberg's e x t r a c t gave values for the p r i n c i p a l reducing substances, to w i t . g l u t a t h i o n e , ascorbic a c i d and -30-cysteine as about 65, 30 and 5 rng. % r e s p e c t i v e l y . These three accounted l o r p r a c t i c a l l y a l l the reducing t i t r e . Ergothioneine was absent (233). B f f e c t of Some Sulphydryl Compounds and Other Reducing Substances on Growth and Development. There was some b a s i s f o r Rowntree's b e l i e f that the constant presence of -5H i n h i s potent e x t r a c t s was more than merely a d v e n t i t i o u s . For s e v e r a l i n v e s t i g a t o r s had shown that sulphydryl compounds stimula, ed c e l l d i v i s i o n (12,103,104,105,106,108,109,166,209,244,286). Evidence was given t h a t s u l p h y d r y l Increased m i t o s i s i n Paramoeceum and Amoeba, that i t increased the r a t e of h e a l i n g of s k i n wounds and t h a t , i n p l a n t s , i t was concentrated i n meristematic regions. Gregory and Davis (93) found that three c o n s i s t e n t l y r e l a t e d f a c t o r s w i t h regard t o r a b b i t s vrere, the adult r a c i a l s i z e , the r a t e of segmentation of the eggs and the concentration of glutathione i n the new born young. With t h i s background and with the knowledge that thymus extract contained such reducing substances as g l u t a t h i o n e , cysteine and ascorbic a c i d , Rowntree et a l . i n v e s t i g a t e d the e f f e c t of administering these substances to r a t s (230). They Injected f r e s h l y prepared s o l u t i o n s of the pure chemicals i n h y d r o c h l o r i c a c i d of the same strength of the extract (pH 4.6) i n d a i l y Ice. i n t r a p e r i t o n e a l doses. With gl u t a t h i o n e alone i t was found necessary to i n j e c t 2mg. d a i l y . But t h i s amount produced very d e f i n i t e p r e c o c i t y even i n the Fg generation. During the f i r s t f o r t y days, the r a t e of growth increased 20^ over the c o n t r o l s . The f i g u r e s they report are as f o l l o w s . -31-Feature Controls Fg generation Birth weight (grams) 4.9 5.2 Average Ears detached (days) 1 . 7 n Teeth erupted (days) 8-10 6.4 it Hair appeared (days) 12-16 10.9 it Eyes opened (days) 14-17 10.5 t< Testes descended (days) 35-40 20.6 it Vagina opened (days) 55-62 34.6 it A dose of 2mg, per day of ascorbic acid was also found necessary - no changes being produced by 0.25 mg. or 1.0 mg. daily. In the second generation no increase in rate of growth was observed but somatic, development was greater than i n the glutathione group. It was most marked in the third generation, the teeth appearing i n 6 days, the eyes opening in 10, while gonadal development was over 50% ahead of the controls. . Cysteine was found to be toxic i n doses of over Jmg. daily. In the offspring of treated rats, though weight was down owing to the toxicity of the solution, yet somatic development was increased appreciably. When glutathione and ascorbic acid were administered together, I mg. of each per day, the offspring of the treated rats were markedly precocious. The figures are, for teeth, 6.2 days; for eyes, 9.6 days; for testes, 16 days; and for vagina, 23 days. Though the effects of these substances did not exactly parallel those of the thymus extracts, yet Rowntree -was quite justified in ascrib-ing; a large part of the latter to glutathione, cysteine and ascorbic acid. Another investigator, Lee, getting his hint from Rowntree's -32-f i r s t work, dosed r a t s with 0.5 to 1.0 mg. of glutathione per day (156). He got unnii stake able p r e c o c i t y i n the f o u r t h generation, the ears opening at I-g- days of age, the eyes at 6-7 days while f o r the gonads, the times are 15-19 days f o r the descent of the t e s t e s and 28 days f o r the opening of the vagina. The above experiments do not seem to have been repeated by any.other worker. Other Reports of the Presence of glutathione and Ascorbic A c i d i n the Thymus. '•'•'he presence of glutathione i n the thymus gland has been reported by others (23,187,280). Schaffer and S l e g l e r (234) have reported the i s o l a t i o n of a small quantity from the thymus. Ascorbic a c i d has also been found i n the gland (23,64,86,130). The amounts reported seem to vary considerably. The Purpose of t h i s Research "When I t became apparent that the attempt by Miss. Dale (see above) to repeat Rowntree's r e s u l t s was not succeeding, a t t e n t i o n was d i r e c t e d towards p o s s i b l e reasons f o r the impotency of the e x t r a c t . The c h i e f of these seemed to be the f a c t that Miss. Dale prepared her e x t r a c t s from the thymi of calves considerably older than thoss by the P h i l a d e l p h i a workers. The l a t t e r had a v a i l a b l e apparently a continuous supply of thymi from calves of 2 t o 6 weeks o l d (258). Hanson wrote that ( i l l i ) " the f i r s t and v i t a l part of the whole process i s to secure healthy thymi from calves under s i x weeks of age". (At t h i s point i t might be mentioned that i n s p i t e of t h i s i n s i s t a n c e on the glands of -33-very young c a l v e s , the glands of correspondingly young r a t s were not used by Einhorn and- Rpwntree (59) i n t h e i r experiments on homologous thymus implants where they obtained d e f i n i t e p r e c o c i t y . The implants were obtained from r a t s 20 to 50 days o l d . The f i g u r e s published show that puberty occurs i n t h e i r untreated r a t s between 4-0 and 60 days of age.) H i s s . Dale, however, was not able-to secure a regular supply of glands of under three months of age. The glands were obtained through the courtesy of Burns and Go.. Since i t i s i l l e g a l i n t h i s province to s e l l meat from calves under t h i s age, younger glands could not be procured. The research reported on below, was undertaken to endeavour to check on any d i f f e r e n c e s i n e x t r a c t s prepared from glands of d i f f e r e n t ages and to compare the r e s u l t s obtained w i t h f a c t o r s mentioned by Rowntree and h i s a s s o c i a t e s . P l a n of I n v e s t i g a t i o n The ?/ork was d i v i d e d i n t o the f o l l o w i n g p a r t s . 1. Determination of the iodine reducing t i t r e of e x t r a c t s prepared by Steinberg's method (258) from glands of d i f f e r e n t ages.. 2. A n a l y s i s of the e x t r a c t f o r g l u t a t h i o n e , cysteine and ascorbic a c i d . 3. Determination of the nitrogen d i s t r i b u t i o n i n the e x t r a c t . Methods of'"analysis f o r g l u t a t h i o n e , cysteine and ascorbic a c i d Glutathione Glutathione was f i r s t i s o l a t e d by Hopkins (126) i n 1921. He showed that i t contained s u l f u r and at f i r s t b e l i e v e d i t to be a. d i p e p t i d s of cysteine and g l u t a n i c a c i d . Hunter.and Eagles (131) threw some doubt on -34-this structure. A reinvestigation by Hopkins (127) i n which he obtained pure crystalline glutathione showed that i t was really a tripeptidfi > containing cysteine, glutamic acid.and glycine. This was soon 00 nf irmed toy Kendal, McKenzie and Mason (145). Glutathione exists xn two forms, an oxidized and reduced form, which have the same relation to each other as cystine to cysteine, being the disulphide and sulphydryl forms respectively. In slightly acid, neutral or basic solution, especially i n the presence of heavy metals, the reduced form rapidly absorbs oxygen becoming converted to the oxidized form. This reaction i s very slow in strongly acid solutions. The f i r s t method put forward for determining the amount of glutathione in biological fluids was Tunncliffe's iodine titration method (279). He used nitroprusside as an external indicator. This procedure was soon modified (79,172,201) by the addition of potassium iodide so that starch could be used as an internal indicator. There have been other variations of the iodine method involving potassium iodate, ferricyanide, . etc. to release iodine from potassium iodide (77,122,189,298).Mason (172) however pointed out that the method which i s excellent for the pure substance may be invalidated by other tissue constituents. Mason, therefore, suggested a new method based upon the oxidation of glutathione by potassium ferricyanide. An equivalent amount of ferrocyanide is formed which i s estimated colorimetrically as Prussian blue (172). This method, i f the pH was carefully controlled, was said to be more specific than the iodine method. Another method of analysis measured the red color which develop when sodium nitroprusside and a l k a l i react with GSH. Bierich and Rosenbohm (22) reported lower values than the iodine method gives. Maloef (167) stated that of a l l t i s s u e c o n s t i t u e n t s only g l u t o t h i o h f i aend cysteine give the r e a c t i o n but Rammett and Chapman (107) found t h a t the method was u n r e l i a b l e f o r q u a n t i t a t i v e estimations. I n 1927 Hunter and Eagles (132) applied a modified form of F o l i n and Looneys c y s t i n e determination (72) to the estimation of glutathione i n pure s o l u t i o n . The method involved comparing the blue c o l o r formed by reducing the s p e c i a l phosphotungstic a c i d reagent i n the presence of sodium'hydroxide with a c y s t i n e standard. The reagent was phospho-I8-t u n g s t i c a c i d (ordinary phosphotungstic a c i d i s phospho-24-tungstic a c i d ) . Shinohara and Padis i n a f u r t h e r study of t n i s method found that g l u t a t h i o n e hydrolysed slowly g i v i n g cysteine and thus caused discrepancies. Glutathione may therefore be estimated as c y s t e i n e , but the two cannot be d i f f e r e n t i a t e d by t h i s method when they are mixed i n s o l u t i o n (251). The method i s thus of l i m i t e d value i n dea l i n g w i t h t i s s u e f l u i d s or e x t r a c t s . Benedict and G o t t s c h a l l (14) used an arsenophosphotungstic a c i d reagent which was reduced by glutathione w i t h the formation of a blue c o l o r . I n 1934 Binet and Weller put forward, a very convenient method I n which reduced glutathione was p r e c i p i t a t e d by cadmium l a c t a t e at a d e f i n i t e pH. The mercaptide so formed was soluble i n a c i d whereupon the G5H was set f r e e and was estimated by a simple iodometric t i t r a t i o n . C a r e f u l pH c o n t r o l was necessary, the p r e c i p i t a t i o n not being complete at a lower pH than 6.8 while an a l k a l i n e medium caused a r a p i d o x i d a t i o n of the g l u t a t h i o n e . Oxidised glutathione could be determined by f i r s t reducing i t with cyanide. Cysteine I n t e r f e r e d w i t h the t e s t . I t was. -36-however, p r e c i p i t a t e d completely at pH 6.4 end thus could be removed p r i o r to the glutathione determination (24,25,26,27). An extremely s p e c i f i c micromethod f o r .the estimation of ^ glutathione depends upon a f u n c t i o n of the l a t t e r as the " a c t i v a t o r " f o r the enzyme, glyoxalase, which converts methylglyoxal to l a c t i c a c i d . As a matter of f a c t the substrate of the enzyme i s not methylglyoxal but a compound formed from methylglyoxal and glutathione, the l a t t e r being set f r e e as a r e s u l t of the enzymic r e a c t i o n . The amount of " a c t i v a t i o n " i s dependent,' w i t h i n a c e r t a i n range, on the concentration of glutathione. The range I s of low concentrations .of g l u t a t h i o n e . Tie enzymic a c t i v i t y was determined by measuring the ra t e at which carbon dioxide was evolved from carbonate by the formation of l a c t i c a cid (Woodward. 297) . A l a t e r m o d i f i c a t i o n was to t i t r a t e the unchanged methylglyoxal w i t h b i s u l f i t e [ (236). Disadvantages of the method would appear to be. the d i f f i c u l t y of g e t t i n g the pure enzyme (a preparation of washed yeast was used), the l i a b i l i t y of the enzyme to poisoning (233) and the d i f f i c u l t y of prepar-i n g methylglyoxal. An enzymic method i s not one which arouses confidence, Quensel and Wachholder put forward an i n d i r e c t method of estimating g l u t a t h i o n e . The t o t a l i o dine reducing t i t r e of the s o l u t i o n was determined before and a f t e r destroying glutathione w i t h d i l u t e formaldehyde (206,287). This procedure was o r i g i n a l l y derived f o r moving a c o r r e c t i o n f o r ascorbic a c i d . The presence of cysteine i n the s o l u t i o n would cause a discrepancy since i t , too, r e a c t s with formaldehyde (124, 164,207) . Another method was based on the r e a c t i o n 2 GSH + S » GoSG 4- HgS -37-The GSH co n t a i n i n g e x t r a c t was digested with elemental s u l f u r In the presence of hydrocyanic a c i d . The hydrogen s u l f i d e formed was determined i o d o m e t r i c a l l y . Any other substance having a f r e e sulphydryl group (notably cysteine) i n t e r f e r e d . Ergothionine d i d not i n t e r f e r e . Results obtained by t h i s method, on po-feioes and l i v e r t i s s u e were lower than those obtained.by simple iodometric t i t r a t i o n and were i n good agreement with those obtained using the Binct and Weller cadmium l a c t a t e p r e c i p i t -a t i o n method (97). • The foregoing review r e v e a l s that only one of the methods suggested f o r the a n a l y s i s - o f g l u t a t h i o n e i s s p e c i f i c f o r that compound That method I s woodward's glyoxalase a c t i v a t i o n method. The other methods depend on the reducing power or some other chemical property s i m i l a r to that possessed by other substances l i k e l y to be present i n b i o l o g i c a l f l u i d s . Cysteine ; U n t i l M ueller i s o l a t e d methionine'in 1922, cysteine with i t s r e v e r s i b l y o x i d i s e d form, c y s t i n e , were the only known natu- a l l y occuring sulfur c o n t a i n i n g amino-acids. As -with g l u t a t h i o n e , the e a r l i e s t method of estimating the concentration of cysteine i n s o l u t i o n s depended on i t s a b i l i t y to reduce Iodine (189,11,160,123,155). But aside from i t s obvious n o n - s p e c i f I c i t y , Buckford and Schoetzow (37) showed that cysteine reacted wi£h iodine i n other ways than to form c y s t i n e as had been p r e v i o u s l y assumed. The formation of a reddish c o l o r w i t h sodium n i t r o p r u s s i d e has been widely used f o r d e t e c t i n g s u l p h y d r y l compounds, but In qua n t i t a t -38-i v e work xt I s subject to a number of sources of e r r o r , the greatest being the I n s t a b i l i t y of the c o l o r (235). The best and most s p e c i f i c c o l o r t e s t f o r cysteine i s that discovered by S u l l i v a n I n 1926 (261). On the a d d i t i o n of sodium I , 2-naphihoquInone-4-sulfonate to a str o n g l y a l k a l i n e s o l u t i o n containing c y s t e i n e , and In the presence of cyanide and s u l f i t e , a reddish brown co l o r develops. This I s converted to a purer red by the a d a l t i o n of sodium hypo s u l f i t e ( H a ^ O^) while any c o l o r given by other substances i s discharged. A large number of other compounds have been test e d and found to be negative while c y s t e i n e alone gives a p o s i t i v e r e a c t i o n (262,264,267, 124). According to S u l l i v a n and Hess (263) the three unsubstituted groups -SH, -NHg and -GOGH are necessary. This c o n d i t i o n i s found only In cysteine and compounds of the same c o n f i g e r a t i o n i n the a l i p h a t i c s e r i e s . The l a t t e r are not known to occur i n animal or vegetable t i s s u e . A m o d i f i c a t i o n of the method was suggested by Lugg (163) who, to maintain the same pH i n assay and standard, swamped both with considerable amounts of g l y c i n e . The o r i g i n a l S u l l i v a n procedure, however, c a l l e d f o r the a d d i t i o n of enough sodium hydroxide to r a i s e the pH very high and Lugg's m o d i f i c a t i o n was s a i d to be much l e s s s e n s i t i v e (266). Based on the discovery that c y s t i n e would reduce the u r i c a c i d reagent of F o l i n and Denis (71) to form a blue s o l u t i o n , F o l i n and Looney developed a qu a n t i t a t i v e t e s t f o r c y s t i n e i n p r o t e i n hydrolysates (72). I'he reagent I s phospho-I8-tungstic a c i d . Cystine i t s e l f i s unaffected by the reagent but In the presence of sodium s u l f i t e i s reduced to cysteine which gives the r e a c t i o n . Many m o d i f i c a t i o n s of t h i s procedure have been made. The -39-method has been studied by Lugg (161,162) and e s p e c i a l l y by Shinohara (245,246,247,248,249,251,252). The l a t t e r w i t h h i s associates thoroughly i n v e s t i g a t e d the nature of the c o l o r r e a c t i o n and developed procedures f o r independently determining c y s t i n e , cysteine and ascorbic a c i d . They were unable to include glutathione i n the scheme (as has already been stated) and t h i s substance i n t e r f e r e d i f present. The methods they devised were accurate though t e d i a u s . A photometric method has also been used (141), Besides the above there have been other procedures put forward from time to time. None have come i n t o widespread use. Fleming (69) (Sound that cysteine reacted with diiaethyl-p-phenylene-diamine hydrochloride when heated i n the presence of a small amount of f e r r i c c h l o r i d e to give a stable deep blue c o l o r . Dyer and Baudisch .(55,13$ reported a degree of s p e c i f i c i t y i n the r e a c t i o n between cysteine and o-benzoquinone, s i m i l a r to that with S u l l i v a n ' s naphthoquinone. % e n an aqueous s o l u t i o n of cysteine was shaken with a chloroform, s o l u t i o n of o-benzoqulnone, a deep red c o l o r developed i n the 'chloroform l a y e r . There are, however, other substances which give the r e a c t i o n or i n h i b i t the c o l o r , i n c l u d i n g glutathione (265). Shinohara and K i l p a t r i c k described a t e s t In which a cobalt complex of cysteine was formed and i t s brownish yellow c o l o r meas-ured In a colorimeter (250). Cysteine has been p r e c i p i t a t e d from a t i s s u e extract w i t h cuprous mercaptide. A f t e r removing the copper, the cysteine was determined by S u l l i v s n s method (231). As o u t l i n e d above, i t i s e a s i l y seen that of a l l the -40-methods put forward f o r the a n a l y s i s of c y s t e i n e , that of S u l l i v a n i s the most s p e c i f i c and has the widest a p p l i c a t i o n . I t i s f o r these very reasons that i t s use has been f a r more widespread than that of any other suggested procedure. Ascorbic a c i d The i s o l a t i o n of the ' antiscorbutic vitamin C was f i r s t reported by Waugh and King (291,292) i n 1932. They i d e n t i f i e d i t with'the "hexuronic a c i d " i s o l a t e d i n 1928 by Szent-Gyorgyi as a t i s s u e r e s p i r a t o r y ' f a c t o r (270). By the above and many other i n v e s t i g a t o r s i t had been recognized that the vitamin was e a s i l y destroyed by mild o x i d a t i o n . I t s s t r u c t u r e was worked out by 1933 and shown to be r — 0 ~ ~ ' H 1 f (65,121,140,182).But before t h i s was established £ q £ £ r (^ff Qfj II i i ( i t had been synthesized by R e i c h s t e i n (208). 0 OH OH Qll xhe e a r l y i s o l a t i o n , synthesis and development of p r a c t i c a l methods of assay have g r e a t l y stimulated the "study of vitamin G so that the l i t e r a t u r e d e a l i n g w i t h i t I s very l a r g e . , - - Before i t s r e l a t i o n s h i p w i t h the known sugars had been worked out Haworth and S z e n t - G y o r g y i suggested the name ascorbic a c i d . The name cevitamic a c i d , has also been used. The f i r s t -reagent f o r estimating the a n t i s c o r b u t i c potency of f o o d s t u f f s was. phosnhotungstomolybdic a c i d , (Mo03(W03)P205(H20)24) introduced by Bezssonoff (18). I t i s u n s a t i s f a c t o r y i n some cases (142) and gives higher values than some l a t e r methods (238). Many other reducing .substances also give the c o l o r . In 1927 i t was reported by Z i l v a t h a t a n t i s c o r b u t i c -4-1-soluiions would rapidly reduce phenoiindophenol to i t s leucobase (301,362). Re considered, t h a t the reducing agent was not the a n t i s c o r b u t i c f a c t o r but that the l a t t e r was s t a b i l i z e d by the former. Re was misled i n t h i s connection by the f a c t that the r e v e r s i b l y o x i d i s e d form of ascorbic a c i d , dehydroascorbic a c i d , i s capable of preventing scurvy but does not reduce Indophenol. That the red u c t i o n of a d e r i v a t i v e of phenolindophenol, 2, . 6-dichlorophenolindophenol, appeared to run p a r a l l e l to the vitamin G content as determined by b i o l o g i c a l assay, was f i r s t reported by T i l l m s n i , R i r s c h and R i r s c h i n 1932 (278). I t was confirmed by H a r r i s -and h i s associated (30,117) and by Bessey and.King (17), Jhe use of t h i s reagent f o r the: a n a l y s i s of ascorbic a c i d has subsequently been very widespread (lo) fhe r e d u c t i o n of the i n d i c a t o r by ascorbic a c i d i s represented i n the f o l l o w i n g equation. HO—C I •I + I HO—c—^H C&0H a A A V I !; A Since t h i s method in v o l v e s the reduction of the indophenol dye by ascorbic a c i d , many other substances having a lower reducing p o t e n t i a l than the reagent are po s s i b l e sources of i n t e r f e r e n c e , i t has been found, however, t h a t most of such substances found n a t u r a l l y react at a-much-slower rat e than ascorbic a c i d which r e a c t s p r a c t i c a l l y Instantaneously (9,17,19,184,288,303). Reduction by phenols, tannins, glutathione' and many other substances was eliminated by t i t r a t i n g i n a c i d -42-s o l u t i o n s of about pE 3 (23). Cysteine however reduces the dye appreciably even at t h i s a c i d i t y , though i t r e a c t s slowercthan does ascorbic a c i d . Unknown reducing m a t e r i a l s are reported to i n t e r f e r e with the t e s t i n nerve t i s s u e (21,299), i n the eye l e n s and aqueous humor (29) and i n cancer t i s s u e s (34,56,116,144). Various substances formed i n heating a l k a l i n e sugar s o l u t i o n s such as reductone and red u c t i c a c i d (17) reduce the dye as r a p i d l y as ascorbic a c i d . Beer, malt and yeast also contain substances which s e r i o u s l y I n t e r f e r e w i t h the t e s t (73,115). Emmerie and van E i k e l e n (62,63) found that some i n t e r f e r i n g substances Including c y s t e i n e , ergothionlne and t h i o s u l f u t e could be removed-with mercuric acetate. I t was necessary to remove mercury wish hydrogen s u l f i d e and then to eliminate excess HgS. The e x t r a steps involved uncertainty and there was danger of l o s i n g some ascorbic a a i d . I n s p i t e of the weaknesses reviewed above and the co n s i d e r a t i o n , Important from the n u t r i t i o n a l atand-polnt, that some of the vitamin might be In r e v e r s i b l y o x i d i s e d form and thus undetectable by indophenol but a v a i l a b l e p h y s i o - l o g i c a l l y , t h i s method has been almost u n i v e r s a l l y adopted. Other suggested methods have been l e s s s p e c i f i c or l e s s s t r a i g h t forward. The indophenol method has been extended to such a micro scale by S l i c k and B i s k i n d using the Linderstom Lang technique that the ascorbic a c i d content of a s i n g l e c e l l may be estimated (33,84,85). A.:.i convenient and ac.curate m o d i f i c a t i o n of the method has been the use of a p h o t o - e l e c t r i c colorimeter to measure the decrease i n co l o r i n t e n s i t y of an excess of the dye on adding the ascorbic a c i d s o l u t i o n (15,68,185). Since the r e a c t i o n of ascorbic a c i d w i t h the dye i s -43-almost instantaneous, a c o r r e c t i o n could be made f o r other reducers by e x t r a p o l a t i n g the reading curve to zero time. Turbid and colored s o l u t i o n s could also be analysed. M e l v i l l e and Richardson (179) suggested using the oxazine, dye, prune, as the i n d i c a t o r . T h e o r e t i c a l l y , i t should be bet t e r than indophenol, since i t represents the l i m i t to which the o x i d i s i n g agent may be weakened and s t i l l permit successful t i t r a t i o n . King (147) however, reported that i t was not as s a t i s f a c t o r y as indophenol. Another a l t e r n a t i v e method i s based on the de c o l o r a t i o n of methylene blue (78) by ascorbic a c i d but t h i s method has not been adopted to any extent. F o l i n * s u r i c a c i d reagent, phospho-18-tungstic a c i d (71) has also been used f o r the estimation of ascorbic a c i d (177,251,252), The i n t e r f e r e n c e of t h i o / compounds ( c y s t e i n e , glutathione etc.) was prevented by the use of formaldehyde (172,177) or mercuric c h l o r i d e (251). ' inis t e s t was s i m i l a r to that of Bezssonoff. Sodium tungstate has also been used, a sky-blue c o l o r developing on i t s reduction (288), but t h i s method has been severely c r i t i c i s e d (285). The a c t i o n of formaldehyde i n destroying i n t e r f e r i n g s u l p h y d r y l compounds i n an iodometric t i t r a t i o n has been used (289). Tauber and K l e i n e r (274) introduced a method In which f e r r i c y a n i d e was reduced and the extent of re d u c t i o n estimated by adding f e r r i c gum g h a t t i reagent and determining the P r u s s i a n blue so formed c o l o r i m e t r i c a l l y . The c o l o r was stable and the reagents were e a s i l y prepared. Glutathione d i d not reduce the ferricyani.de I f the pH i s l e s s _44-than 2 "but tannins and other phenols and cysteine d i d reduce i t . Spent-Gyorgyi (270) found t h a t the adrensj. cortex reduced s i l v e r n i t r a t e and ascribed t h i s property to i t s vitamin G content. Harde (113) developed an a n a l y t i c method on t h i s r e a c t i o n but i t s value has been questioned, some i n v e s t i g a t o r s considering i t useless (31,269, 285). Giroud and Leblond (81) found t h a t a p o s i t i v e t e s t was very s p e c i f i c f o r ascorbic a c i d , but that a negative t e s t did not prove i t s absence owing to the presence of f a c t o r s i n h i b i t i n g the reduction. The dark v i o l e t c o l o r of a ferrous-ascorbic a c i d complex has been used i n a c o l o r i m e t r i c t e s t by Szent-Gyorgyi (272). The formation of the complex was sa i d to depend on the -HOC : COR- grouping. Scudi and R a t i s h (238) based a method on the reduction of d i a z o t i z e d s u l f a n i l a m i d e i n a c i d s o l u t i o n . They claimed t h a t cysteine d i d not i n t e r f e r e . I t has been reported by Sevine (243). t h a t the reduction i n the col d of an a c i d i f i e d s o l u t i o n of sodium s e l e n i t e to free selenium was a s p e c i f i c r e a c t i o n of ascorbic a c i d . Roe (219,220) has described a method whereby ascorbic a c i d was converted by b o i l i n g w i t h h y d r o c h l o r i c a c i d Into f u r f u r a l . The l a t t e r was determined w i t h a n i l i n e . I t i s doubtful i f t h i s procedure can be standard-i z e d to give reproducible r e s u l t s . I n 1930 Szent-Gyorgyi (27l);< obtained an enzyme from cabbage capable of o x i d i s i n g "hexuronic a c i d " . Tauber, K l e i n e r and Mishkind (275) prepared a s i m i l a r o x i d a t i v e enzyme from the p e r i c a r p of squash which they claimed was s p e c i f i c f o r ascorbic a c i d . Others have also prepared t h i s enzyme (153,257), which has been c a l l e d ascorbic a c i d oxidase. I t s -45-I t s a p p l i c a t i o n to the a n a l y s i s of ascorbic a c i d involved iodine or indophenol t i t r a t i o n s before said a f t e r t r e a t i n g a s o l u t i o n with the enzyme (257,275). Doubt, however, has been thrown on the s p e c i f i c i t y of the enzyme (137,255). The enzyme has not been obtained i n the pure form and i t has been claimed t h a t the ox i d a t i o n of ascorbic a c i d was due to the c a t a l y t i c a c t i o n of t r a c e s of copper (43,128,146,259,250). A spectroscopic method Involved measuring the i n t e n s i t y of the absorption band of ascorbic a c i d at 265 mu.. I'he extent of in t e r f e r e n c e was estimated'by destroying the ac i d by the a d d i t i o n of copper s a l t s and remeasuring the band (218). Another p h y s i c a l method f o r use i n cases where extraneous reducers were absent was the a d d i t i o n of c c p p e n i o n s to a s o l u t i o n i n which ascorbic a c i d was being e l e c t r o l y s e d using a dropping mercury ano€e. A polarographic "wave" appeared i n the current voltage curve at -1.60 v o l t s (from a calomel zero)(149). A l l the methods proposed for the' determination of ascorbic acid have various disadvantages. Of the methods f o r which s p e c i f i c i t y was claimed, the enzymic procedure I s u n c e r t a i n , the spectroscopic and polarographic r e q u i r e expensive equipment. I n the other, chemical t e s t s , at l e a s t c y s t e i n e , i f not other substances a l s o , i n t e r f e r e s by reducing the reagents us.ed. I t i s probable t h a t a completely s p e c i f i c method cannot be based on the reducing powers' of ascorbic a c i d . But of those which are so based, and which, as has been seen, Includes p r a c t i c a l l y a l l , there i s none so s t r a i g h t forward and so simple as Tillman's 2, 6-dichlorophenolin-dophenol method. I t i s rat h e r strange that i n a.decade of research, no other method has been devised which can compare with regard to ease and accuracy w i t h a t e s t introduced e a r l y In vitamin G research before the -46-vitamin had been I s o l a t e d or any of i t s p h y s i c a l or chemical properties e s t a b l i s h e d . -47-EXPER.IMSNTAL WORK I . P r e p a r a t i o n of the E x t r a c t Thymus glands of calves were obtained as soon as p o s s i b l e a f t e r k i l l i n g and were placed, on "dry i c e " u n t i l ready to be extracted. They were then allowed to p a r t l y thaw out, were cut i n t o small pieces w i t h a c h i s e l and put through a meat gr i n d e r . The t i s s u e was weighed and placed i n a I l i t e r round-bottomed f l a s k . An amount of 1-/4 h y d r o c h l o r i c a c i d was poured over i t so that there was 1.0 cc. of a c i d f o r each 1,5 g r . of gland. The mixture was s t i r r e d i n the c o l d f o r about 10 minutes a f t e r which the temperature was r a i s e d r a p i d l y to 68 G, s t i r r i n g the while with a s t i r r e r run by a small e l e c t r i c motor. The mixture was then cooled with running water. The f l a s k and i t s contents were l e f t overnight i n the cooler to a l l o w the greater part of the sediment -settle on the bottom. The e x t r a c t was f i l t e r e d o f f through a Buchner funnel using s u c t i o n . I t was kept i n pyrex rubber-stoppered f l a s k s in.the c o o l e r , 0.2/i c h l o r b u t a n o l being added as a p r e s e r v a t i v e . Determination of the t o t a l I.R.T. A lOcc. p o r t i o n of e x t r a c t was a c i d i f i e d with 0.5cc. concent*-,-'.'./ t r a t e d h y d r o c h l o r i c a c i d , a f t e r which IOcc. of I0/o phosphotungstic a c i d were added dropwise with s t i r r i n g . The mixture was cooled f o r 10-15 minutes. The p r e c i p i t a t e was f i l t e r e d o f f and washed thoroughly with ifc h y d r o c h l o r i c a c i d , the washings being caught i n the f i l t r a t e . I c e . of f r e s h 5% s o l u t i o n and 6 drops of 5r/a starch s o l u t i o n _ 4 3 -were added to the f i l t r a t e . 0.001 N ICIQ^ s o l u t i o n was added In excess from a b u r e t t e . The blue c o l o r so formed ?,Tas discharged with standard 0.001 M Na2S203 s o l u t i o n and f u r t h e r KIC13 was run i n to the reappearance of a blue t i n t , stable f o r one minute. The i . r . t . was c a l c u l a t e d as mg.% g l u t a t h i o n e , by the f o l l o w i n g formula, 1. R.T. s V x M xM x 100 E where V i s the t o t a l volume of KI.O3 used l e s s the equivalent of the NagSgOs added, K i s the normality of the K I O 3 , M i s the molecular weight of glutathione (307), and E i s the volume of extract used. The I.R.T.s of e x t r a c t s prepared from glands of calves of varying ages were measured and are tabulated below.(page 48a) 2. A n a l y s i s f o r Glutathione I . Formaldehyde method G r i f f i n (94) t e n t a t i v e l y used Wachholder's method which Involved d e s t r u c t i o n of glutathione by formaldehyde, and comparing the i o d i n e reducing value of the s o l u t i o n with a sample not so t r e a t e d with formaldehyde. The a p p l i c a b i l i t y of t h i s method was checked by determining i t s e f f e c t s on cysteine and ascorbic a c i d . . Cysteine was destroyed completely a f t e r standing one hour i n a s o l u t i o n , c o n t a i n i n g about 4y£ formaldehyde. The l o s s of a s c o r b i c a c i d under s i m i l a r c o nditions was found to be 31.7% as compared with a c o n t r o l t i t r a t i o n . TABLE SHOWING VARIATION IN I.R.T, IN EXTRACTS FROM GLANDS OF' DIFFERENT AGES AGE FETAL 2-8 months WEEKS 4 5 6 MONTHS 2 3 4 MONTHS 6 8 10 YEARS 2 I.R.T* Mgf0: GSH 114 165 158 \% 181 245 l8.0# 215 205 242 230 150# 143# 248 243 209 19,8 219 224 169# 138# 269 l8o## Average I.R.T i n the different age groups 155 . 219 202 199 193 & This series of extracts with low i . r . t , values were made up at one time and had been treated s i m i l a r l y . 7^ 7/These glands were much involuted. The thymic tissue had to be separated from much f a t and connective tissue. - 4 9 -The method o u t l i n e d f o r 'determining the t o t a l i odine reducing t i t r e -could not be employed because of the r e a c t i o n between t h i o s u l f a t e and formaldehyde. The end-point was therefore the appearance of the blue c o l o r onorunning i n the standard K I O 3 s o l u t i o n the f i r s t time. 2. Cadmium l a c t a t e p r e c i p i t a t i o n method The B i n e t and w'eller method which p r e c i p i t a t e d glutathione as a cadmium l a c t a t e mercaptide was employed. Reagents T r i c h l o r a c e t i c a c i d a. 10% s o l u t i o n ' Metaphosphoric a c i d a5% f r e s h l y prepared s o l u t i o n Cadmium l a c t a t e a saturated s o l u t i o n . The cadmium l a c t a t e was prepared by d i s s o l v i n g Gadmium hydroxide (from cadmium s u l f a t e and sodium hydroxide) i n l a c t i c a c i d . The s o l u t i o n was evaporated and cooled t o c r y s t a l l i z e out'the cadmium l a c t a t e . I t was d i s s o l v e d i n water and r e e r y s t a l i i z e d once. The c r y s t a l s were-then thoroughly washed with acetone u n t i l the washing l i q u i d was n e u t r a l . Sodium hydroxide 0.5N and Q.lN s o l u t i o n s Phosphoric a c i d a 10% s o l u t i o n Procedure ' To 5cc. of e x t r a c t , 5c.c. of 10% t r i c h l o r a c e t i c a c i d was added. A f t e r s t i r r i n g and c o o l i n g , the p r o t e i n p r e c i p i t a t e was centrifuged down. One c.c. of 5/£ metapho sphoric a c i d was added, followed by 2c .c. of cadmium l a c t a t e s o l u t i o n (jf) . §• I f t o t a l g l utathione ( o x i d i s e d as w e l l as reduced) was to be determined I c e . of f r e s h 5% potassium cyanide s o l u t i o n was added to the deproteinized -50-The solution was neutralized to ph 5.fci (not over 7.0) and allowed to stand overnight. A white precipitate formed immediately. This precipitate was centrifuged down, the supernatent was discarded, the precipitate was washed with alcohol and dissolved i n lu% phosphoric acid. The glutathione content was determined by iodometric titration i n the manner earlier described. Interference of cysteine Binet and Weller removed cysteine by a preliminary precip-itation at pH 6.4 i n spite of the fact that glutathione had started to precipitate at this point. The loss of glutathione after three hours standing at pH 6.4 was estimated by comparing the iodine reducing t i t r e of the precipitate, calculated as cysteine, with the cysteine content of the extract as determined, by Sullivan*s method ( see page 51). Table.showing cysteine content i f extract estimated by different methods. Extract 5:Cysteine content by Sullivan's Method Ug% (Average). Cysteine Content by Cd Lactate Method Mg/£ (Average). Reduced Glutathione Content by Cd Lactate Method after ppting. cysteine. Mg/S (Average) : i . 5.4 7.0 19.2 2. 7.0 7.8 12.9 3. 5.2 6.4 14.4 extract which was then allowed to stand for thirty minutes before adding metaphosphoric acid and proceeding with the determination. -51-Because of t h i s l o s s of glutathione I n the cysteine p r e c i p i t a t e , both substances were brought down together at a pH of 6.3. To. get the glutathione content of the e x t r a c t the equivalent of the cysteine content found was subtracted from the t o t a l value obtained. Since pure glutathione was not a v a i l a b l e or e a s i l y prepared, the method has not been checked. 3. A n a l y s i s f o r Cysteine The procedure adopted was the s p e c i f i c S u l l i v a n c o l o r i m e t r i c " method. The standardized procedure c l o s e l y f o l l o w s that proposed by S u l l i v a n . Apparatus Colorimeter The instrument used was a Klett-Summersoil photo-e l e c t r i c c o l o r i m e t e r , made by the K l e t t Manufacturing Company of Hew York. Reagents. Sodium cyanide. A I/i s o l u t i o n i n 0.8N sodium hydroxide. This s o l u t i o n I s stable f o r several weeks.. Sodium s u l f i t e . A IQ/o s o l u t i o n (anhydrous s a l t ) i n 0.5H sodium hydroxide. This s o l u t i o n i s stable f o r months. Sodium hypo s u l f i t e (or hydro s u l f i t e ) (MagSgO^SRgO). A 2% s o l u t i o n i n 0.5M sodium hydroxide. This s o l u t i o n must be f r e s h l y prepared a few minutes before u s i n g . Sodium hydroxide. A 5K s o l u t i o n . I , 2-Haphthoquinone-4—sodium su l f o n a t e . A I/i aqueous s o l u t i o n , f r e s h l y prepared. -52-BttgCBxaire" ;. • To 5cc. of the t e s t s o l u t i o n i n a colorimeter tube the f o l l o w i n g reagents were added i n order. The contents of the tube were mixed a f t e r each a d d i t i o n by i n v e r t i n g the tube. F i r s t , 2c.c. of sodium c y a i i d e s o l u t i o n , followed by I c e . of the naphthoquinone reagent and f i n a l l y 5c.c. of sodium s u l f i t e s o l u t i o n . The tube was then allowed to stand f o r t h i r t y minutes at a temperatwre of 20-25°G. In the case of pure s o l u t i o n s of cy s t e i n e , the s o l u t i o n was then d i l u t e d w i t h 2c.c. water, and i n the case of the e x t r a c t , with 2c.c. 5H sodium hydroxide to get a high a l k a l i n i t y i n the h i g h l y buffered s o l u t i o n . At the end of the t h i r t y minute period. I c e . of sodium h y p o s u l f i t e . s o l u t i o n was added and mixed. The tube was l e f t to.stand f o r ten minutes before the c o l o r i n t e n s i t y sas measured i n the colorimeter. This t e n minute p e r i o d ¥/as found necessary to permit the c o l o r to become f a i r l y s t a b l e . The cysteinecontent of the s o l u t i o n was read o f f a c o l o r i n t e n s i t y - . -c o ncentration graph prepared using s o l u t i o n s of knov/n concentrations of cys t e i n e hydrochloride i n 1/t hydr o c h l o r i c a c i d . The graph i s herewith Included. When the naphthoquinone was added, the s o l u t i o n unless i t was a pure s o l u t i o n of c y s t e i n e , turned a d i r t y brown or black c o l o r . The ad d i t i o n of sodium h y p o s u l f i t e removed t h i s dark c o l o r l e a v i n g the s o l u t i o n a pure red c o l o r , or orange i n very d i l u t e s o l u t i o n s of c y s t e i n e . Extent of i n t e r f e r e n c e by ascorbic a c i d To determine whether or no ascorbic a c i d i n t e r f e r e d w i t h the zO.<— -53-development of the c o l o r , varying amounts of i t were added to s o l u t i o n s of known cy s t e i n e content, 'which were then t r e a t e d by S u l l i v a n ' s method. A brown s o l u t i o n r e s u l t e d on the a d d i t i o n of naphthoquinone. This was apparently completely cleared by the h y p o s u l f i t e leaving" only the b r i g h t red caused by c y s t e i n e . Tab leashowing recovery of cysteine from s o l u t i o n s containing ascorbic a c i d . Cysteine Content Mg. /5 c.c. Ascorbic A c i d Added Mg. /5 c.c. Cysteine determined Mg. /5 c.c.(Average) Percentage Recovery % 0.15 0.5 0.17 113 §>.I5 1,0 0,18 120 0.15 1.5 ; 0.18 120 0.15 2.0 . 0.17. 113 ;.o.,i5' • • - 2.5 0.175 116 0,15 3.0 0.145 96 0.25 0.5 0.24 96 0.25 1.0 0.24 36 • .0.25' 1.5 '. 0.26 104 0.25 2.0 0.25 100' 0.25 2.5 0.24 96 0.25 3.0 0.23 92 Recovery of cy s t e i n e added to e x t r a c t I n order t o t e s t the a p p l i c a b i l i t y of the method to thymus e x t r a c t , c ysteine was added to p o r t i o n s of the e x t r a c t . I t s cysteine content was determined before and a f t e r such a d d i t i o n . -54-Tabls showing recovery of cysteine added to thymus e x t r a c t . Cysteine content' Cysteine C o l o r - Cysteine Recovery of 1 Percental of e x t r a c t . added imeter Determined ' added cysteine Recovery Eg./5c.c. (Average) Mg./5c,c. Reading Mg./5c.c. Mg./5c.c. % 6. I I 0.06. , 80 0.165 0.055 92 0.11 0.06 82 0.175 0.065. 108 •0.10 O.H 93 0,225 0.125 . 114 0.10 . o . i i .92 0.22 0.12 109 4. A n a l y s i s f o r Ascorbic A c i d The d i r e c t t i t r a t i o n of an ascorbic a c i d s o l u t i o n w i t h a standard s o l u t i o n of 2, 6 dichlorophen.olindoph.enol or, conversely, the t i t r a t i o n of a measured quantity o i the dye w i t h the ascorbic a c i d s o l u t i o n both of which methods have been widely used, was found to be u n s a t i s f a c t o r y from the p o i n t of view of accurate estimation. I n the f i r s t p l a c e , the end-point was very vague owing to the weak c o l o r and to the formation of brownish substances as a r e s u l t of the r e a c t i o n betireen the indophenol and the a c i d . No concentration of dye or of ascorbic a c i d would give a s a t i s f a c t o r y end-point. I n the second place, the time taken f o r the t i t r a t i o n would increase the e f f e c t of i n t e r f e r i n g substances, e s p e c i a l l y c y s t e i n e , i f they were present, as would be the case i n t i t r a t i o n of the e x t r a c t . Recourse was therefore made to a procedure i n v o l v i n g the use of a p h o t o e l e c t r i c c o l o r i m e t e r . In t h i s method, v i s u a l t i t r a t i o n was replaced by an o b j e c t i v e p h o t o e l e c t r i c measurement of the amount of dye decolorized when a measured amount of an ascorbic a c i d s o l u t i o n reacted with an excess of dye, the i n i t i a l depth of c o l o r of the dye being measured before hand. Since greater, d i l u t i o n s were used, the brown r e a c t i o n products did not become troublesome and. In f a c t , could not be detected. Also the determination could be ef f e c t e d i n ten to f i f t e e n seconds, thus minimizing e r r o r s due to the presence of other dje reducing substances. Since s t r o n g l y g.cid s o l u t i o n s d e c o l o r i z e the dye to a s l i g h t extent, the readings were made i n a sodium acetate b u f f e r so that the pH was 3.0 to 3.5. This pH was not low enough to cause s i g n i f i c a n t f a d i n g of the dye and yet was not so high as to increase s e r i o u s l y the rate at which other reducing substances react w i t h the dye. Apparatus The p h o t o e l e c t r i c colorimeter used was the K l e t t p r e v i o u s l y described. A 9c.c, r a p i d - d e l i v e r y p i p e t t e made by c u t t i n g the lower end of f a IOc.c. p i p e t t e and r e c a l i b r a t i n g to d e l i v e r 9 c . c . Reagents. 2, 6-dichlorophenolindophenol s o l u t i o n , approximately 0.05 gr. per 100 c.c. of water. This s o l u t i o n was f i l t e r e d . I t would keep several weeks i f stored In a dark g l a s s b o t t l e . D i l u t i o n of t h i s s o l u t i o n IsIO r e g i s t e r e d about 150 on the colorimeter i n a blank determination using I c e . of 1% h y d r o c h l o r i c a c i d . Sodium acetate. A s o l u t i o n of 10 gr. of NaCgH'sO^'SHgO i n 100 c.c. of water was made up. A few drops of toluene were added to ensure i t s keeping. Dye-acetate s o l u t i o n . The indophenol so d i l u t e d and mixed that 9c.c, of the dye-acetate s o l u t i o n and I c e . of an a c i d s o l u t i o n containing 0,5^ H C 1 and 2.5^ H P 0 3 gave a reading of about 150 on the colorimeter and had a pH of 3.0-3.5. Ascorbic a c i d s o l u t i o n s of known concentration i n Ij£ H G 1 . Metapho sphoric a c i d . A f r e s h l y prepared 5,-= s o l u t i o n . Hydrochloric a c i d . A 1% s o l u t i o n . procedure The galvanometer of the colorimeter was set at zero using a tube f i l l e d w i t h d i s t i l l e d water($). The. i n i t i a l o p t i c a l d ensity of the dye was determined by'placing I c c . of an a c i d mixture of Q.5% H G 1 and 2,5% H P O 3 i n a colorimeter tube, running i n 9c.c.. of the dye-acetate s o l u t i o n , i n v e r t i n g the tube once immediately p l a c i n g the tube i n the c o l o r i m e t e r and t a k i n g the reading. \'f The colorimeter must have been switched on .and allov/ed to warm up not l e s s than an hour before making the determination. I t must also be protected from draughts, bunsen burners e t c . , since a temperature change would a f f e c t the reading. Iff The r a p i d a d d i t i o n of 9c.c. of the dye-acetate s o l u t i o n to I c . c . of test s o l u t i o n was supposed to ensure complete-raising. Some d i f f i c u l t i e s exper-ienced at f i r s t were found to a r i s e from incomplete mixture, v a r i a t i o n s In the c o l o r , o f t e n h a r d l y d i s t i n g u i s h a b l e , occuring from one part of the tube to another. The f i n a l procedure therefore c a l l e d f o r i n v e r s i o n of the tube p r i o r to t a k i n g the reading. A tube co n t a i n i n g I c . c . of te&t s o l u t i o n and 9 c . c . d i s t i l l e d water was placed i n the colorimeter and the l i g h t f i l t e r was adjusted f o r any c o l o r or t u r b i d i t y i n the s o l u t i o n , the galvanometer needle being brought back to zero. The f i n a l o p t i c a l density was then determined i n the same manner as abote, the reading being made as soon as p o s s i b l e . The r e l a t i o n s h i p between the amount of dye decolorized and the concentration of ascorbic a c i d was given by the f o l l o w i n g equation. X — K ( L]_ — ) where X i s the concentration of ascorbic a c i d L-^  i s the i n i t i a l o p t i c a l density of the dye L 0 i s the f i n a l o p t i c a l density of the dye, and K i s a constant, depending on the type of colorimeter-u.nd the •q u a n t i t i e s of reagents used. Determination of K ' The ascorbic a c i d s o l u t i o n s i n I/d HG1 were d i l u t e d with an equal' volume of 5% H P O 3 . This f i n a l concentration of ascorbic a c i d i s X. (see table page 58). A p p l i c a t i o n of the method to the a n a l y s i s of thymus -extract. I, P r e c i p i t a t i o n of p r o t e i n . The f o l l o w i n g p r o t e i n p r e c i p i t a n t s were investiget ed, t r i c h l o r -a c e t i c a c i d , t a n n i c a c i d , metaphosphoric a c i d and phosphotungstic a c i d . (I) T r i c h l o r a c e t i c a c i d slowly faded the dye and, at concentrations necessary f o r p r o t e i n p r e c i p i t a t i o n , sbout 5%, was found to destroy ascorbic a c i d . -58-Table showing valises of K determined X i n mg. % H (ir) L 2 (#) K 3.955 151 125 0.15 6.59 . 151 106 0.T5 7.325 151 103 Q.I5 144 90 - 0.15 10.99 151 77, 0.14 113.56 - 144 39 0.13 14.65 151 34 0.125 15.40 130 17 0.14 16.45 144 7 0.1.2 §, These values are averages of hot l e s s than three determinations Average value f o r K i s 0.14. ( i i ) Tannic a c i d reduced the dye very r a p i d l y . ( i l l ) Metaphosphoric a c i d d i d not a f f e c t e i t h e r the dye or the ascorbic a c i d . I t has, i n f a c t , been recommended f o r s t a b i l i z i n g the l a t t e r (188). The ord i n a r y commercial metapho sphoric a c i d was found to be a very I n e f f i c i e n t p r o t e i n p r e c i p i t a n t , so i t was t r e a t e d a f t e r F o l i n (70) as follows? 200 grams of commercial metaphosphoric were fused with 2 grams of potassium n i t r a t e i n a c r u c i b l e . Heating was continued u n t i l a l l water had been d r i v e n o f f and fumes of phosphorus pentoxide began to appear. The s l a g was skimmed o f f , and the melt was poured i n t o t i n p l a t e s ( I t stuck' f a s t to p S r c e l a i n ) and Yihen c o o l was broken Into pieces and stored i n a t i g h t l y stoppered b o t t l e . The glassy product had to be ground very f i n e ol,.i since i t d i s s o l v e d w i t h d i f f i c u l t y . ( i v ) Phosphotungstic a c i d d i d not a f f e c t the dye and appeared not to a f f e c t the ascorbic a c i d though a blue c o l o r developed i n the s o l u t i o n when i t was mixed w i t h the l a t t e r . (3) Recovery of ascorbic a c i d added to e x t r a c t : A p o r t i o n of e x t r a c t was deproteinized by adding dropwise an equal volume of e i t h e r lKffa phosphotungstic a c i d or 5% metophosphoric a c i d . A f t e r s t i r r i n g and c o o l i n g the p r e c i p i t a t e was centrlfuged down. One c.c. p o r t i o n s of the supernalent s o l u t i o n were taken f o r t e s t i n g , ;.' To another p o r t i o n of the'same e x t r a c t , ascorbic a c i d was added. I t was then deproteinized l i k e the foregoing. .. Table showing recovery of ascorbic a c i d added to extract Gone. Mg./o i n E x t r a c t (Aver age) Ain't Mg.% added P r o t e i n Pptant. Used Amt. Recovered (Aver age) Percentage Recovery 4.2 35,6 Pho sphotungstic 40,3 I0I.4/o 15.6 22,4 ttetapho sphoric 37.6 97.5^ 15,, 6 13,9 i i 30.7 105.1/o (2) Extent of i n t e r f e r e n c e by c y s t e i n e . Cysteine a l s o reduces the. indophenol reagent, and there i s a small amount of cyst-aine I n the e x t r a c t . Cysteine hydrochloride was t h e r e f o r e added to a deproteinized e x t r a c t to determine the extent to which i t would i n t e r f e r e under the c o n d i t i o n s of t h i s procedure. -60-Tabie showing rocovery of ascorbic a c i d from cysteine-enriched extract Ascorbic Acid'Content Cysteine Ascorbic A c i d Percentage before adding cysteine added Mg./'o Recovered Mgjt Recovery Mg./£ (Average.). 8.5 5.0 8.3 97 © G^o 8.5- • 9.5 7.3 35. T/o The amounts of cysteine added were equal or greater than the amounts probably already i n the s o l u t i o n . E r r o r s caused by the reduction of the dye by cysteine are not serious i n t h i s procedure. Conclusion . ' - .,.. The method o u t l i n e d above f o r the estimation of ascorbic a c i d •would seem to give r e s u l t s of . f a i r accuracy when applied to thymus e x t r a c t . 5. A n a l y s i s of Thymus E x t r a c t Two po r t i o n s of the ex t r a c t were deproteinlzed, each w i t h an equal volume, i n one case, of 5% metaphosphoric a c i d , and i n the other, of 10/o t r i c h l o r a c e t i c a c i d . Ascorbic a c i d , "cysteineand the t o t a l I.R.T. were determined i n the metapho sphoric a c i d f i l t r a t e and glutathione In the t r i c h l o r a c e t i c a c i d f i l t r a t e . The r e s u l t s are tabulated i n the f o l l o w i n g t a b l e , !{ page 61) 6. Determination of the Nitrogen d i s t r i b u t i o n -The p r o t e i n content was determined by a K j e l d a h l method, the nit r o g e n content being measured before and a f t e r p r e c i p i t a t i n g the p r o t e i n (119). -TABLE SHOWING CONCENTRATIONS AND I.R.T. PIT REDUCING CONSTITUENTS OF THYMUS EXTRACT Ascorbic. Acid Glutathione Cysteine Sum of Reduction of three Constituents ' i Extract Total I.R.T. Mg $ GSH Concen-tration Mgfo Percent . of Total Reduction i Concentra-t i o n Mgfo Percent of Total Reduction i Concen-tr a t i o n Mgfo Percent of Total Reduction i 1 208 : / l 3 7 . 2 6 2 . 6 3 1 . 9 24 .95 7 .6 9.1 2 2C!4 9 9 . 2 6 7 . 2 3 6 2 . 3 ' 3 0 . 5 7 .0 8 . 6 1 0 6 . 3 5 3 ; -.174. 2 8 . 8 3 7 . 9 4 7 - 9 2 7 . 5 7.4 '..'10.6 9 6 . Q' 4 178 2 7 . 4 3 3 . 9 6 7 . 3 ; 37*8 6 . 0 9 . 0 1 0 0 . 7 3 20? - 34/2 ' 3 8 . 6 , 71*35 3 4 . 1 6 . 8 8 . 3 1 0 1 . 0 6 198 2 6 . 8 : 47.4 6 0 . 7 5 3 0 . 7 6.4 . 8 . 6 8 6 . 7 7 219 3 6 . 8 5 8 . 7 7 0 . 5 3 2 . 2 6.4 7 . 3 9 8 . 2 Average : 1 9 8 . 6 3 2 . 9 : 3 7 . 9 61 .7 3 1 . 1 6 . 8 8 .6 97*6 To 10c.c. of extract or f i l t r a t e i n a E j e l d a h l f l a s k , 20c.c. concentrated s u l f u r i c a c i d and 0.2 gr. copper s u l f a t e were added. The mixture was heated u n t i l i t became a l i g h t green, the fumes being removed by s u c t i o n through a funnel over the mouth of the f l a s k . A f t e r a l l o w i n g the f l a s k to c o o l , 200 c c of water were added, followed by concentrated sodium hydrosB.de u n t i l the mixture was a l k a l i n e . The f l a s k was cooled during t h i s a d d i t i o n . Several pieces of granular zinc were added to.prevent bumping, and a small piece of p a r a f f i n t o check foaming. The contents of the f l a s k were d i s t i l l e d over Into standard s u l f u r i c a c i d , , a K j e l d a h l t r a p being i n s e r t e d to prevent splashing over of any a l k a l i . Excess s u l f u r i c a c i d was t i t r a t e d with standard sodium hydroxide s o l u t i o n using methyl orange as i n d i c a t o r . The p r o t e i n p r e c i p i t a n t s used were metaphosphoric a c i d and phosphotungstic a c i d . . A l l determinations were done i n d u p l i c a t e and average values were taken. Table showing d i s t r i b u t i o n of Nitrogen i n Thymus e x t r a c t . T o t a l Nitrogen 0.31 Gr. /lOOc.c . E x t r a s i Ifon P r o t e i n Nitrogen H P O 3 F i l t r a t e 0.29 ti 11 i t P r o t e i n Nitrogen 0.02 ti » 11 Nitrogen i n Phosphotungstic A c i d F i l t r a t e 0.07 11 11 11 Nitrogen i n Phosphotungstic A c i d P r e c i p i t a t e 0.24 11 11 11 -63-DISGUSSION • This investigation was for the purpose of checking on any chemical difference between Miss Dale's extract and that prepared by Rowntree and his associates, which might account for the difference i n biological effects. Unfortunately there were but few cr i t e r i a to use i n making the comparison. There were as follows, (l) the iodine reducing powere of the extract, (2) the absolute content of, and the distribution of the reducing power between, the three reducing substances, glutathione, ascorbic acid and cysteine, and (3) the total nitrogen and the protein nitrogen content of the extract. Before discussion these factors I would emphasize the fact that extracts made up by somewhat different methods have been reported as biologically active. Hanson, like ourselves, made use of solid carbon dioxide to preserve the glands from autolysis until the extract was to be made up. Rowntree, on the other hand, immediately ground the fresh gland into cold 1% acid which was then transported to the laboratory. With regard to the actual extraction, Hanson's original method involved a slow heating to 94-96°C while Steinberg's called for a rapid heating to 68°C. Hanson at f i r s t used menthol as a preservative. He wrote in 1938 that Rowntree used no preservative at a l l . Steinberg's recipe included the addition of chlorbutanol. The only variation used by us and not reported as used by others was to leave the extraction mixture in the ice box overnight i n order to f a c i l i t a t e f i l t r a t i o n . The only essential difference between our extracts and those of the other workers would appear to be the age of the calves used, -64-and this difference on f i r s t sight would not appear, to be important. For calves at 3-4 months of age are s t i l l well removed from sexual maturity (over 12 months). Their thymi ( to judge by analogy with human beings and rats) are probably s t i l l growing though at a slower rate relative to the growth of the animal than those of 2-6 weeks old calves. (Here one may recall the observation made above concerning the age of rats from which thymi were taken for the implanting experiments). However there was the possibility that the difference in age might show i t s e l f i n some chemical difference i n the extract. A series of extracts was therefore made up from glands of varying ages and the results are tabulated on page 48a. Inspection of these reveals that there i s a slight decrease i n I. R. T. with an increase in the age of the gland. It may be questioned whether the variations are significant and i n my opinion they are not so. It i s seen that there i s great variation i n the values i n each age group. Though the 4-6 weeks group had the highest average, the 6-10 months group (average about 9% less that 4-6 weeks) contained the highest value recorded. The t i t r e of the f u l l grown steers i s of similar magnitude to the rest. Its slightly lower average i s probably due to the time consumed in freeing remaining glandular tissue from large amounts of connective tissue and fat. It is probable that the I. R. T. of any-'animals thymus i s not a function of i t s age. The second criterion was the amounts of the three reducing constituents. The results of my analyses of extracts of 3-4 months old thymi are i n the table on page 61. These results with their comparative constancy and with the closeness to which the sum of the percentages of the total I. R. T. of the determined amounts of glutathione, cysteine and ascorbic acid approach 100% are very gratifying. They are also similar to values reported from 2-6 weeks-old-thymus extracts by Schaffer, Ziegler and Rowntree. For convenience i n comparing the two sets ofresults, the following table i s included ( page 66). It gives the average values found by myself and the averages given i n each of Schaffer, Ziegler and Rowntree's two tables. Since the latter include the values found in extracts which had aged up to 20 days, there are also tabulated the values found i n four extracts which are more comparable with mine, whose analyses were complete within four days of the making up of the extracts. Bearing i n mind, great variations which may occur i n the I. R, T. of extracts prepared i n identrical manner from different glands considerable (Variations i n the content of the substances responsible for that variation would be expected. But the significant fact i s that the percentage of the total I. R. T, of any one of the three substances deter-mined l i e s within one same range both i n Schaffer's results and in mine. This would indicate that the composition of the gland at the two ages i s , i n regard to iodine reducing substances capable of being set free by the conditions of extraction, very similar. I do not believe that any other conclusions may be drawn. Tt i s of great interest to note that Miss Dale's rats were receiving somewhat over half a milligram of glutathione daily. Now this is the amount of glutathione, withwhich Lee, as reported earlier, obtained an unmistakeable precocity of growth and development i n the fourth consecutive generation of injected rats. It w i l l also be remembered, however, that Rowntree could get similar precocity only with 2 mg. per day, TABU COMPARING THE CONCENTRATIONS AND I.R.T.. OF 'THE REDUCING CONSTITUENTS .OF THxW,SrEXTRACTS : Ascorbic Acid Glut a t h i oris Cysteine Sum of Reduction of three Constituents i . Extract Total I.R.T. Mg % GSH Concen-tration Mgf. Percent of t o t a l Reduction % Concentra-t i o n Mgfo Percent of Total Reduction i Concen-trat i o n Mgfo Percent-of Total Reduction 1 Average (Smith) 1 9 8 . 6 3 2 . 9 3 7 . 9 6 1 . 7 / . 3 1 . 1 6 . 8 8 . 6 (a)Average (S.,Z.&R.) 149 2 3 . 0 5 7 . 8 4 3 . 3 2 8 . 9 6 . 4 ; L 4 . 3 1 0 1 . 2 ("b) Average (S^Z.&R.) 152 2 1 . 3 3 1 . 9 ; 3 0 . 8 40 .7 3 . 1 3 . 3 9 8 . 1 Extracts (S.,Z.&R.) 1 . ( 1 day) 179 3 0 . 3 5 7 . 7 6 3 . 7 3 6 . 7 2 . 8 3 . 0 9 9 . 4 2 . ( 9 days) 109 1 8 . 3 : 3 8 . 3 2 6 . 3 24.2 * 7 .9 ?3.3 1 0 6 . 0 3 . ( 1 day) 188 31 .'.0 • 3 4 . 1 7 0 . 9 37 .7 4 . 6 6 .2 9 8 . 0 4 . ( 6 days) 171 2 7 . 1 3 2 . 0 38.7 ' 3 4 . 3 3 . 9 8 .7 9 3 . 0 -67-a result which may have been connected with the purity of his preparation. However when 1 mg. of ascorbic acid was also given, 1 mg. of glutathione was a l l that was required. Miss Dale's rats received somewhat less than a third of a milligram of ascorbic acid daily. The failure of Miss Dale's rats to respond even i n the f i f t h generation increases the doubt with which one contemplates the results claimed by the group of investigators working around Rowntree. With regard to the nitrogen content, Steinberg reported his to contain 0.43 grams per 100 cc. The protein content was practically negligible. My figures show ( see page 62) that though the total nitrogen was only 0.31 grams /£, 0.02 grams % were precipitated by metaphosphoric acid and are tabulated as protein nitrogen. This larger protein content may have been due to the practise of leaving the extract in contact with the tissue for up to twenty four hours. This larger amount of protein i s the only difference of any magnitude between the factors reported of Rowntree's extracts and ofthose used here. The very large precentage of the nitrogen precipitated by phosphotungstic acid shows that a large pact of i t exists as various degradation products of proteins. -68-C.ONCLUSIONS' 1« The I. R. T's. of thymus extracts have been determined, the content i n these extracts of glutathione, ascorbic acid and cysteine have been estimated and the nitrogen distribution i n the extracts has been indicated. 2. The values so obtained have been compared with values reported by L. G. Rowntree and his co-workers for the extracts they used. No differences i n the composition of the extracts could be found which would account for the impotency of the one and the potency of the other to cause accumulative increase i n the growth and development of succeeding generations of rats. BIBLIOGRAPHY 1 . Achertj J.E. and M.H. M o r r i s Studies on the e f f e c t of thymectomy on growing chickens; Anat. Record 44, 209. 1929. 2. A l l a r d y c e , J . , U. D a l e , D. Smith and P. G r i f f i n F a ctors i n potency of thymus ez t r a c t s s Endocrinology 27, 994. 1940. 3. A l l e n , B.M. The r e s u l t s of the e a r l y removal of thymus glands from Rana pi p i e n s tadpoles? J . Exp. Zool. 30, 189. 1920. 4. A l l e n , E. The r e a c t i o n s of immature monkeys to the i n j e c t i o n of ovarian hormones J . Morph. P h y s i o l . 46, 479. 1928. 5. iindersen, D.H-, "_. :• The r e l a t i o n s h i p between the thymus and reproductions P h y s i o l . Rev. 12, I . 1932 6. Andersen D.H. Studies on the physiology of reproductions I . 'fhe e f f e c t of thymectomy and season i n the age of puberty i n the female r a t i J . P h y s i o l . 74, 49. 1932. I I . E f f e c t of thymectomy on the age of puberty i n the male rats 74, 207. I I I . E f f e c t of thymectomy on f e r t i l i t y i n the r a t : 74, 212. 7. Arey, L.B. Developmental Anatomyj W.B. Saunders Company, P h i l a d e l p h i a and London. 1934. 8. Asher, L. and P. R a t t i On the e f f e c t s of administering thymocresin : Endocrinolggy 15, 467 „ 1931. 9. Bacharach, A.L. and H.T. Glynn Determination of ascorbic acids Nature 136, 757. 1935. 10. Badertscher, J.A. The development of the thymus i n the pigs Am. J . Anat. 17, 437. 1915 11. Baernstein, H.D. ' -Gasometric determination of cysteine and cystines J . B i o l . Chem. 89, 125, 1930. 12. Baker, L.E.' The chemical nature of substances required f o r c e l l m u l t i p l i c a t i o n . 2. The a c t i o n of g l u t a t h i o n e , haemoglobin and ash of l i v e r on;,the growth of f i b r o b l a s t s : J . ^ xp. Med. 49, 163. 1929. 13. Baudlsch, 0,,^. and^E^Dyer The o-quinone t e s t f o r c y s t e i n e ; J . B i o l . @hem. 99, 485. 1933. 14. :? Benedict,,. 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