It is suggested that insulin in blood circulates in two forms : a biologically active free form and a biologically inactive form which we called bound insulin or insulin complex. The properties of free insulin appear to be similar to those of crystalline insulin extracted from pancreas. Bound insulin, on the other hand, exhibits different physicochemical characteristics and is unreactive with anti-insulin antisera. Partially purified preparations of bound insulin have been obtained from human or animal sera by resin adsorption and elution. These preparations exhibit about a 300-fold protein purification. Further purification about 3,000-fold) can be achieved by Sephadex chromatography of these preparations.
Bound insulin preparations produced in vivo and in vivo biologic effects similar to those of crystalline insulin. The biologic effects of bound insulin have been studied on practically all the in vivo and in vivo systems available for the detection of small amounts of insulin. Since there is no evidence that substances other than insulin can produce the sum of these in vivo and in vivo effects of bound insulin we assumed that bound insulin activity represent true insulin activity.
Bound insulin may represent a metabolite of free insulin which is produced in vivo by the action of extrapancreatic tissues. Bound insulin is not generated in vitro by the addition of crystalline insulin in sera. Recent studies suggest that liver perfusion with crystalline insulin results in the production of bound insulin-like substances with properties similar to those of serum bound insulin. This is consistent with the suggestion that the liver may play an important role in the transformation of free to bound insulin. Total pancreatectomy in rats produced a significant decline in bound insulin concentrations.
Studies in nondiabetics suggest the possibility that insulin activity may be regulated through a dynamic balance between active, free and inactive, bound insulin. A malfunction of the biochemical mechanisms which regulate this balance at the tissue level may represent one of the primary lesions in the pathogenesis of some types of diabetes. Such a malfunction may cause increased transformation of free to bound insulin by extrapancreatic tissues and/or a decline in the rates of activation of bound insulin by the tissues of these patients. Inactive bound insulin, therefore, accumulates in the blood of these diabetics. This high concentration of bound insulin, in turn, may interfere with the normal clearance and utilization of the endogenous free or exogenous crystalline insulin.
In vivo studies indicate that adipose tissue extracts (ATE) can produce a significant decline in the blood glucose concentrations of adrenalectomized and hypophysectomized rats. Similar effects with ATE were produced in intact, spontaneously diabetic mice of the KK strain. The effect of ATE in these diabetic animals lasted 24 to 30 hours. The KK mice are considered to represent an inbred diabetic strain with features characterizing human diabetes of the maturity-onset type. It is suggested that the effect of ATE in these animals may result from the
In vivo activation by ATE of their circulating inactive bound insulin.

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Using the technique of the isolated rat diaphragm to measure insulin activity and antagonism, our initial studies on untreated or uncontrolled insulin-requiring deabetic patients showed that their plasma inhibited the activity of insulin in vitoro (Vallance-Owen, Hurlock & Please, 1955). It was then found that this antagonism of insulin was associated with the albumin fraction of the plasma proteins (Vallance-Owen, Dennes & Campbell, 1958a). Whole plasma from normal subjects and from obese maturity-onset diabetics, who ordinarily do not require insulin therapy, has no measurable insulin antagonism. Nevertheless, when normal plasma is broken down into its various constituents, insulin antagonism can be detected in the albumin fraction, although it is less active than the corresponding fraction prepared from the plasma of the insulin-requiring diabetics.
When tested at 3.5-5.5%, both diabetic and normal albumin completely inhibited the effect of 1000 micro-units/ml of insulin added in vitoro. At 1.25%, however, the diabetic albumin was still highly antagonistic, whereas albumin from normal subjects was inactive.
This antagonistic activity appears to depend upon both the pituitary gland and the adrenal steroids (Vallance-Owen, Dennes & Campbell, 1958b Vallance-Owen & Lilley, 1961) it is not due to the albumin itself but to some substance associated with it, hence the term synalbumin antagonist.
It has also been found that albumin from obese maturity-onset diabetics and from latent diabetics is antagonistic to insulin when tested at 1.25% to the same extent as albumin prepared from insulin-requiring diabetic patients, and to a considerably greater degree than normal albumin, which is inactive at this concentration. Moreover, patients with the diabetic syndrome of carbohydrate intolerance, but suffering from definite pancreatic disease such as acute pancreatitis or haemochromatosis or who have sustained total pancreatectomy, have no increased antagonism to insulin associated with their plasma albunim (Vallance-Owen, 1962, 1964).
Thus essential diabetics, whether insulin-requeing, obese or in the latent phase, have more synalbumin antagonism than normal subjects or pancreatic diabetics. These observations indicate that excessive synalbumin antagonism (synalbumin-positive) can be regarded as a biochemical marker to ascertain whether or not a given individual is constituted as an essential diabetic-Without reference to carbohydrate intolerance. On this premise the presumed genetic transmission of essential diabetics has been examined by studying the relatives of patients suffering from this condition.
The results strongly suggest that excessive synalbumin antagonism is inherited as an autosomal 'dominant' character. Ninety-seven members of nine families were studied, thirty-nine were synalbumin-negative whereas fiftyeight were synalbumin-positive, but only sixteen of these latter have overt carbohydrate intolerance. A further three have spontaneous hypoglycaemia whereas the remainder are quite asymptomatic. These observations suggest that overt carbohydrate intolerance is relatively uncommon or will be a late event in many people constituted as essential diabetics (Vallance-Owen, 1966).
Recently a number of studies have been made on the albumin prepared from cord blood of synalbumin-negative mothers and of synalbuminpositive mothers, who were either in the latent or overt phase of diabetes. Twentyseven observations were made and the results are shown in Table 1.
It can be seen that synalbumin-positive mothers have cord bloods which are negative as well as positive. Therefore, it is clear that a child at birth can either be synalbuminpositive (constituted as a diabetic) or synalbumin-negative (normal).

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Although insulin was discovered in 1921, diabetes mellitus is still on the increase. There is no doubt but that absolute or relative insufficiency of insulin causes diabetes mellitus. From this viewpoint ILA is most important.
We wish to find out at what point insulin loses its effect by measuring it with three methods at the same time : diaphragm (DILA), fat pad (FILA) and radioimmunoassay (IRI).

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Recent endocrinological knowledge on proteohormone producing tumor has drawn attention to the comparison of the hormone secreted from normal cells with that from tumor cells. But, reports on a comparative study of the biochemical character of gonadotropins from normal chorionic tissue and chorionic tumor tissue are still scarce.
In the present study, HCGs from chorionic tumor and normal pregnancy were purified by Sephadex gelfiltration and ion-exchange cellulose column chromatography. For a strict comparison between these HCGs, identical procedures were employed for the purification of every preparation. Urinary gonadotropin was extracted by alcohol precipitation method and Kaolin-Adsorption method. Biological activity of HCG was assayed by ovarian weight method referring to 2nd international standard HCG. Immunological activity was assayed by hemoagglutination inhibition reaction.
1) Comparison of HCGs on CM-C column chromatography.
Both HCGs were eluted in two fractions on Sephadex gelfiltration. The retarded fractions were biologically inactive but the unretarded fractions were biologically active. These unretarded fractions were further separated into non-adsorbed part and adsorbed part on CM-C column chromatography. Only the non-adsorbed part was biologically active in both HCGs.
This is an interesting difference from the pituitary gonadotropins, since biological activity of pituitary gonadotropin is observed in the adsorbed part but not in the non-adsorbed part when it is fractionated on CM-C column chromatography.
2) Comparison of HCGs on DEAE-C column chromatography.
In both HCGs, biological activity was present in adsorbed fraction but not in nonadsorbed fraction. In other words, the biologically active protein is adsorbed on DEAE-C but not adsorbed on CM-C. The activity was as high as 20,000 IU/mg in the preparation from normal pregnancy and 8,000 IU/mg from chorionic tumor patient.
When HCG activity of adsorbed protein was seen in connection with conductivity, most of the HCG activity from normal pregnancy was eluted between 3.0 and 5.0 m mho, but that from chorionic tumor was eluted in two parts at more than 5.0 and less than 2.0 m mho but it was not eluted at the conductivity between 3.0 and 5.0 m mho at which normal HCG activity was eluted. On the other hand, when such biologically active adsorbed protein was subjected to linear gradient elution, the amount of eluted protein from normal pregnancy showed a monophasic peak, whereas, the amount of protein from chorionic HCG was much larger at very low and high conductivity at which a very little amount of the protein from normal pregnancy was eluted.
These facts suggest that chorionic tumor HCG is composed of two kinds of protein (acidic and basic) with complexed constitution which differs from that of normal HCG, and they further suggest the difference of the biochemical character of both HCGs.
Recovery of biological activity and the amount of protein were calculated as follows. In normal pregnancy, recovery ratio of protein was 42.5% by CM-C column chromatography and 9.8% by the final DEAE-C column chromatography as to the fraction having 20,000 IU/mg of biological activity, the recovery ratio of protein was 2.2% and that of biological activity was about 50%. In chorionic tumor HCG, the recovery of protein. was 30.6% by CM-C column chromatography and 4.3% by DEAE-C column chromatography, but final recovery of biological activity was 42%.
In both HCGs, immunological activity was high in the fractions with low conductivity. In such fractions of normal pregnancy, both activities were almost parallel, but they were not so in chorionic tumor HCG. And, in general, the fractions having high biological activity were eluted at high conductivity and showed very low immunological activity. Such tendency of dissociation of biological activity and immunological activity was more evident in chorionic tumor HCG,

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Characteristic behavior of the chorionic tumor was investigated from (1) estimation of gonadotropic activity in chorionic tumor patients and (2) estimation of nucleic acid (DNA, RNA) content of chorionic tumor tissue, and the results were compared with those of normal pregnancy.
As chorionic gonadotropin, which is excreted in large amounts by pregnant women and patients with chorionic tumor, is believed to be a proteohormone, chorionic gonadotropin producing mechanism was examined in correlation with nucleic acid content in trophoblastic cells of normal pregnancy and chorionic tumor tissue, because nucleic acid, especially RNA, is a reliable indicator of protein synthesis.
Chorionic gonadotropin content in urine, serum and trophoblastic cells of pregnant women at various gestational stages were studied through the method of bioassay (ovarian weight method of immature female rats) in comparison with those in urine, serum and chorionic tumor tissue of patients with chorionic lesion.
Nucleic acid (DNA, RNA) content in trophoblastic tissue in normal pregnancy and chorionic tumor tissue was assayed chemically with the Schmidt-Thannhauser-Schneider technique.
The following results were obtained showing that the concentration of chorionic gonadotropin in urine, serum and trophoblastic tissue of pregnant women was very high in the second and third months and then it declined abruptly and remained low for the remaining time of pregnancy; and chorionic tumor patients excreted chorionic gonadotropin in a large amount, as large or more than the early stages of normal pregnancy.
In the case of normal chorionic tissue, RNA content was increased in early pregnancy and RNA/DNA ratio was higher than those of any other stage of pregnancy, which indicated the activated RNA synthesis. From these findings, a possibility of close relationship was suggested between the increased chorionic gonadotropin production and the activated RNA synthesis at this stage of pregnancy.
In chorionic tumor tissue, absolute amount of DNA and RNA did not differ too far from that of normal chorionic tissue in early pregnancy, but RNA/DNA ratio was lower than in normal chorionic tissue. This fact suggested that there was a difference between normal pregnancy and chorionic tumor as to biosynthesis of protein, that is chorionic gonadotropin.

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In order to make clear the ovarian function in chorionic tumor patients, estrogen and pregnanediol content in urine of these patients was estimated with the Shida-Kambegawa technique. Further, in chorionic tumor tissue and trophoblasts in normal pregnancy, the activity of Δ⁵-3β-hydroxysteroid dehydrogenase, an enzyme required in the metabolic pathway from dehydroepiandrosterone to Δ⁴-androstenedione and from pregnenolone to progesterone, was stained histochemically with the Wattenberg technique. Ovaries and tumor tissue of these patients were studied histologically and the results of hormaonal assay were compared with them.
Steroidgenetic ability of chorionic tumor tissue estimated by its Δ⁵-3β-hydroxysteroid dehydrogenase was remarkably lower in chorionepithelioma than in normal pregnancy, which suggested that estrogen or pregnanediol excreted in the urine of patients was not produced in the chorionic tissue as in normal pregnancy, but origiated from the ovary of the host. However, urinary estrogen and pregnanediol value of these patients is low in spite of the high amount of chorionic gonadotropin.
This apparently unexplainable discrepancy can be ascribable to the ovarian function of the host. When excessively secreted gonadotropin acts on the ovary, which is its target organ, the lutein cells are activated in the early phase, but gradually regress, decreasing urinary estrogen and pregnanediol. As the disease progresses, dissociation between increased gonadotropin and decreased estrogen arid pregnanediol become pronounced.

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Either estradiol benzoate (E.B.) or progesterone (Prog.) was injected iv. in single doses or ip. repeatedly for 10 days to each group of 1) normal adult female rats and 2) immature female rats, and their effects on the gonadal-stimulating activity of anterior pituitary were studied.
Estimation of the fresh weight and gonadotropic potency (mouse uterine weight) of the anterior pituitary as the criteria of its gonadal-stimulating function and of the ovarian fresh weight, its ascorbic acid concentration, its total- and ester-cholesterol concentration were carried out in order to know the output intensity of gonadotropin form the anterior pituitary.
The result are summarized as follows :
1) The experiments on normal adult female rats.
Gonadotropic potency increased immediately after iv. single doses of 100γ of E.B. indicating the acceleration of anterior pituitary function. And at the same time, ovarian function was activated after the same injection, increasing its weight and decreasing its ascorbic acid concentration, its total- and ester-cholesterol concentration (Table 3, Fig.4). Whereas gonadotropic potency failed to respond, though anterior pituitary weight markedly increased, the ovarian function was reduced as the result of ip. 10 days' repeated injec-tion of 50γ of E.B. (Table 4, Fig. 5).
In the case of ip. 10 days' repeated injections of 5 mg. of Prog., gonadotropic potency and anterior pituitary weight increased tcmporarily during the adoministration, and at the same time, the ovarian ascorbic acid concentration as well as its ester-cholesterol concentration significantly increased, indicating the reduction of the ovarian function (Table 5, Fig. 6).
2) The experiments on immature female rats.
In order to avoid the influence of the endogenous ovarian sex steroids on the effect mechanism of exogenously administered steroids such as E.B. or Prog. of the anterior pituitary gonadal axis, the immature female rats, which had no active ovarian functions at that time, were utilized in these experimental studies. The immature female rats were observed to have a greater pituitary gonadotropic potency than adult females have in the diestrous stage, and to have almost the same concentration of ovarian ascorbic acid. (Table 6, Fig. 8).
Anterior pituitary function as well as ovarian function was markedly increased at 12-36 hours after iv. single doses of 20γ of E.B. (Table 7, Fig. 9). Almost the same results were observed at 12-36 hours after iv. single doses of 2 mg. of Prog. with the exception that the anterior pituitary weight failed to increase in this case (Table 8, Fig. 10).
Anterior pituitary function and ovarian function were reduced from the beginning of ip. 10 days' repeated administrations of 20γ of E.B., continuously to the 5th day after the end of treatments (Table 9, Fig. 11).
The result in the case where 2 mg. of Prog. was injected ip. daily for 10 days was almost similar to the former case except that anterior pituitary weight was not significantly altered, although it markedly increased in the former (Table 10, Fig. 12).
From the above results, it is indicated firstly that the response pattern of anterior pituitary to administered sex steroid such as E.B. or Prog., supposing the fashion of their administration is the same, is very similar and is not dependent upon the difference of endocrinological conditions of the animals. For example, when E.B. is injected iv. in single doses, anterior pituitary gonadal-stimulating function and consequently the ovarian function are activated in the adult female rats as well as immature female rats.
Secondly, the same sex steroid affects the anterior pituitary function in an almost opposite pattern when it is administered either iv. in single doses or successively, being indifferent to the endocrinological condition of the animals. For example, when E.B. is injected iv. in single doses to the adult female rats,

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In the first report, it was observed that both estradiol benzoate (E.B.) and progesterone (Prog.) activate the anterior pituitary ovarian function of normal adult female rats and of immature female rats when all of them are administreed iv. with single doses. And that on the contrary, they reduce their function when administered ip. repeatedly for 10 days. And it was also recognized that the effects of sex steroids on the anterior pituitary gonnadalstimulating activity are quite indifferent both from the endocrinological conditions of the animals and from the kind of steroids such as E.B. and Prog.
In this paper, either E.B. or Prog. was administered iv. in single doses or ip. repeatedly for 10 days separately to each of 3) ovariectomized adult female rats and 4) intrasplenic ovarian autotransplanted gonadectomized female rats, and their effects on the gonadal-stimulating activity of anterior pituitary were studied, especially from the histological findings of intrasplenic autotransplanted ovaries, the alterations of gonadotropin output of the anterior pituitary are discussed.
Estimation of the anterior pituitary fresh weight and gonadotropic potency (mouse uterine weight) as the criteria of its gonadal-stimulating function and histological findings of intrasplenic autotransplanted ovaries, which were stained with Sudan III, was referred to know gonadotropin output intensity from the anterior pituitary.
The results are summarized as follows :
3) The experiments on gonadectomized adult female rats.
On the 10th day after gonadectomy of the adult female rats, anterior pituitary weight and gonadotropic potency started to increase and extended the tendency of their increasing constantly over 80 days after the operation (Table 11, Fig. 14).
Gonadotropic potency remarkably increased, whereas anterior pituitary weight remained unchanged as the result of iv. single-dose injection of 100γ of E.B., which was performed on 30th day after the ovariectomy (Table 12, Fig. 15). Anterior pituitary weight of ovariectomized female rats which were injected 50γ of E.B. daily ip. for 10 days, increased during the whole period of administration, whereas gonadotropic potency of the same animals reduced from the 5th day to the end of successive injections (Table 13, Fig. 16). In the case where 5 mg. of Prog. were injected ip. successively for 10 days, gonadotropic potency decreased after the injection period was finished (Table 14, Fig. 17).
4) The experiments on intrasplenic ovarian autotransplanted adult female rats.
Ovariectomy on both sides was performed, and at the same time one of them was autotransplanted into the spleen of the adult female rats; anterior pituitary started to increase the weight and gonadotropic potency immediately after the operation and was followed by constant rise extending over 40 days after, almost like the case of ovariectomized female rats (Table 15, Fig. 19).
When 100γ of E.B. injected iv. in single doses on the 20th day after the operation, anteior pituitary function was observed to increase temporarily at 36 hours after the injection (Table 16, Fig. 20). On the other hand, intrasplenic autotransplanted ovarium was completely occupied with active corpora lutea, indicating the elevation of gonadotropin, especially of LH output from the anterior pituitary.
As the result of daily successive administrations of 50r of E.B. for 10 days, although the anterior pituitary weight remarkably elevated, gonadotropic potency reduced in the later half of the period of administration, indicative of reduction of anterior pituitary function (Table 17, Fig. 21); and in addition, the histological findings of the intrasplenic ovarium being abundant with sudanophilic granules both in corpora lutea and in interstitial cells, designated the inhibition of gonadotropin output from the anterior pituitary. In the case of daily successive administration of 5 mg. of Prog. for 10 days,

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Several methods have been used for the assay of insulin in human serum. The bioassay methods most commonly used utilize the insulin sensitivity of the gulcose uptake by isolated rat diaphragm, or the oxidation of glucose by the isolated rat epididymal fat pad. More recently, immunochemical techniques have been widely used, which depend on competition between serum insulin and I¹³¹-insulin for a limited amount of guinea-pig antiinsulin antibody. There are considerable differences in the results between bioassay and radioimmunoassay. Furthermore, these results have different significances. Pathogenesis of diabetes mellitus was studied by determination of serum insulin concentration in normal and untreated diabetic subjects before and after glucose loading. Serum insulin concentrations were determined using the rat diaphragm assay (DILA), the rat epididymal fat pad assay (FILA) and the radioimmunoassay technique (IRI) for the same serum samples and at the same time. Twenty-three subjects with no known diabetic heredity served as controls. All of them had normal glucose tolerance. Forth-five patients with diabetes mellitus except Kimmelsties-Wilson's syndrome had never received insulin or oral hypoglycemic agents. They were classified according to fasting blood sugar levels as either severe diabetic group with that above 200 mg/dl or mild diabetic group with that below 199 mg/dl. Ten chemical diabetics were contained in mild diabetic group. Mean fasting levels of DILA, FILA and IRI in normal subjects were 310±50 μu / ml, 399±148 μu ml and 21.3±7.3 μu ml ; in mild diabetic group 203±74 μu / ml, 531±92 μu/m1 and 32.8±9.6 μu/ml; in severe diabetic group 100±22 276±45 μu ml and 9.3±1.9 μu ml, respectively. These observations indicate that fasting DILA levels are lowered in both diabetic groups, and fasting FILA and IRI levels are elevated in the mild diabetic group. But both FILA and IRI in severe diabetic group were lowered. Blood sugar, DILA, FILA and IRI were measured in serum obtained from normal and diabetic subjects in the fasting state, and thirty, sixty, ninety and one hundred and twenty minutes following 50 g oral glucose loading. In normal subjects, DILA, FILA and IRI rose briskly, reaching a peak within thirty to sixty minutes, and then declined gradually. Its mean peak levels of DILA, FILA and IRI were 673±114 μu/ml, 821±168 μu ml and 78.0±13.2 μu/ml, respectively. In the mild diabetic group, the initial FILA was definitely delayed but its concentrations continued to rise for ninety minutes, eventually reaching average levels of FILA considerably higher than that achieved at any time in normal subjects, and the initial IRI was definitely delayed to ninety minutes. The mean peak levels of FILA and IRI were 1402±166, μu/ml and 79.6±14.8 μu/ml at ninety minutes after glucose loading. However, peak responses of DILA in mild and severe diabetic groups to glucose loading were lowered and that in the severe diabetic group were more delayed than in normal subjects. Peak levels of DILA in mild and severe diabetic groups were 373±96 μu/ml and 181±40 μu/ml respectively. Peak responses of FILA and IRI in the severe diabetic group were more lowered and more delayed than in normal subjects. Mean peak levels of FILA and IRI in the severe diabetic group were 371±242 μu/ml and 17.7±8.0 μu/ml at ninety minutes after glucose loading.
The pattern of insulin response of chemical diabetes was very similar to that of the mild diabetic group. The observation shows diminished insulin effect to muscle tissues and suggests the possibility that the serum of mild diabetic subjects contains a large amount of ineffective insulin to muscle tissue but of effective insulin to fat tissue.
Therefore,

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It has been well established that the majority of corticosteroids and their metabolites in blood is usually excreted as a water soluble glucuronide into urine. However, there have been strong evidences to indicate that some part of corticosteroids can conjugate with substances other than glucuronic acid.
In order to elucidate the types of corticosteroid conjugates, the various methods for the extraction and subsequent separation of 17-OHCS-conjugates in urine were fundamentally investigated. Experiments were first made to obtain the optimum condition under which 17-OHCS glucuronides were completely hydrolysed and second to separate and quantitate the non-glucuronide-conjugates. The results were summarized as follows :
1) A modified method for the determination of urinary total 17-OHCS, proposed initially by Glenn and Nelson was reinvestigated mainly on the amounts of beta-glucuronidase and the incubation period neccessary for the complete hydrolysis of glucuronides, and a proper organic solvent and its volume for the quantitative extraction of hydrolysed corticosteroids.
It was ascertained that 20000 F.U. of beta-glucuronidase and 30-40 ml of chloro-form per 10 ml of urine were satisfactory for the determination of the urinary 17-OHCS. 2) Definite evidence was presented for the existence of corticosteroid conjugates in urine, which could not be split with beta-glucuronidase, but were cleaved by the solvolysis procedure with acidified ethyl acetate. The thin layer chromatography of the solvolysate of these conjugates suggested that they are sulfated corticosteroids and these sulfoconjugates were estimated to be approximately 8.0 per cent of the total Porter-Silber chromogens in urine.
3) Three extraction methods for conjugated coticosteroids, with ethanol-ether (1 : 3), with ethyl acetate, and with charcoal-celite were compared.
In the model experiment using the authentic cortisol-21-sulfate, ethanol-ether extraction method was superior to the charcoal-celite method for the recovery of the conjugate from urine. But when the former method was applied to the extraction of conjugates from urine, the intensity of “background” color in the Porter-Silber reaction was considerably high and the extract was not applicable for the high voltage paper electrophoresis as a final step for the separation of conjugates. On the other hand the intensity of “background” in the charcoal-celite method was relatively low and the method was useful for the extraction of conjugates from a large amount of urine. Moreover, this method is recommendable as a rapid routine method for the determination of urinary Porter-Silber chromogens.
4) In a separate series of experiments using column chromatography, it was demonstrated that cortisol, cortisol-sulfate and the pyridinium salt of cortisol-sulfate can be separated by a column of either Sephadex G-25 or LH-20. The pyridinium salt of cortisol-sulfate was eluted in the earliest fraction of any of these columns, but the elution order of cortisol and cortisol-sulfate in a Sephadex G-25 column showed the reverse of that in a LH-20 column. The recovery of authentic free and conjugated steroids from these columns was proved to be almost 100 per cent.
5) High voltage paper electrophoretic analysis of the conjugate extract obtained from urine after administration of 4-C¹⁴-cortisol, preliminarily purified by Sephadex G-25 column chromatography, revealed four radioactive peaks which consisted of glucuronide, mono-sulfate, disulfate and unknown-conjugates.
On electrophoresis run at pH 2.2 the disulfate traveled toward the anode faster than the monosulfate, and the glucuronide stayed at the application line of the paper strip. By the scintillation counting of radioactivities in the individual peak the ratio of these conjugates was tentatively given.

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