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Diurnal rhythm of plasma immunoreactive corticotropin-releasing factor in normal subjects
Life Sciences, Vol. 40, pp. 1651-1655 Printed in the U.S.A.
Pergamon Journals
DIURNAL RHYTHM OF PLASMA IM~4UNOREACTIVE CORTICOTRf~IN-N~.FASING FACTOR IN NORMAL SUBJECTS Toshio Watabe*, Koshi Tanaka**, M~nehito Kumagae*, Sachiko Itoh*, Mitsutoshi Hasegawa**, Toshiyuki Horiuchi**, Syuji Miyabe**, HidetoOhno*** and Naokata Shimizu* Third Department of Medicine*, First Department of Medicine**, Teikyo University School of Medicine, 2-11-1, Kaga, Itabashi-ku, Tokyo 173, Japan and Central Research Laboratory***, Mitsubish Petrochemical Co. Ltd., 1315, Ami-machi, Inashiki-gun, Ibaraki-ken, 300-03, Japan. (Received in final form February 2, 1987)
summary Plasma corticotropin-releasing factor (CRF), corticotropin (ACTH) and cortisol levels were simultaneously determined by radioimmunoassays at 0600h, 1200h, 1800h and 2200h in six normal subjects, in order to examine whether the diurnal rhythm in plasma CRF exists and how it correlates to the diurnal rhythm in plasma ACTH and cortisol concentration. The highest CRF level was observed at 0600h (7.0 ± 1.2 pg/ml) and significantly lower levels (p<0.01) at 1800h (1.7 ± 0.2 pg/ml) and 2200h (1.9 ± 0.4 pg/ml). A clear diurnal rhythm was demonstrated in plasma ACTH and cortisol levels, with the highest values at 0600h (44.6 ± 8.1 pg/ml and 15.9 ± 2.0 ~g/dl, respectively) and the lowest at 2200h (12.3 ± 2.8 pg/ml and 4.6 ± 1.0 ~g/ml, respectively). These results suggest that the diurnal rhythm in ACTH and cortisol is under the regulation, at least in part, of the diurnal rhythm in CRF secretion. It has been well documented that there exists a diurnal rhythm in secretion of adrenocorticotropic hormone (ACTH) (i, 2) as well as other proopiomelanocortin (POMC)-relatedpeptides, such as B-lipotropin (i, 2) and B-endorphin (3). The release of ACTH and POMC-relatedpeptides from the pituitary, including diurnal variation, is assumed to be controlled mainly by corticotropin-releasing factor (CRF), although diurnal rhythm in CRF secretion in man has not been proven as yet. The amino acid sequence of human CRF (hCRF) was deduced recently from the nucleotide sequence of cloned DNA (4), following the isolation of CRF from ovine hypothalami by Vale et. al. (5). The availability of synthetic replicate of hCRF has made it possible to measure hCRF by radioimmunoassay (RIA) in various tissues (6) and in plasma (7). In the present study, imm~noreactive CRF (IR-CRF) was measured in plasma of normal subjects at various times of the day to examine whether there is a circadian variation in the plasma CRF concentration and how it correlates to those in plasma ACTH and cortisol.
0024-3205/87 53.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
1652
Diurnal Rhythm in Plasma CRF in Man
Vol. 40, No. 17, 1987
Materials and M~thods Six normal male subjects were studied after informed consent was obtained from each subject. They were aged from 23 to 31 (average 26.0) years old a n d w e i g h e d from 55 to 70 (average 62.2) kg. The subjects participated in this study while maintaining their customary daily routines of activity and meals throughout the study period. Blood specimens were obtained at 0600h, 1200h, 1800h and 2200h from an antecubital vein into a tube containing EDTA3K. Plasma was separated at 4°C by centrifugation and stored at -20°C until assay. Plasma IR-CRF was measured by a specific RIA after extraction of 20 ml plasma with immunoaffinity chromatography. CRF antiserum was partially purified by precipitation of -~-globulin with 50 % saturated ammonium sulfate and dialyzed. The 3"-globulin fraction was further purified with DEAE cellulose column, lyophilized and coupled to activated CH Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden) as reported (8). A portion (2.0 ml) of the antibody-coupled Sepharose 4B was packed in a small column and 20 ml of sample plasma was applied. The column was washed with 3.0 ml of 0.05 M phosphate buffer pH 7.4, containing 0.45 % NaCI and 0.i % Tween 80, followed by 3.0 ml of distilled water. The retained CRF was eluted with 2.5 ml of IN acetic acid, lyophilized and reconstitated with 0.7 ml of RIA buffer just prior to assay. Synthetic CRF standards or r e c o n s t i t u t ~ s a m p l e s were incubated with antiserum (final dilution 1:1,000,000) and I--D-CRF in a total volume of 0.5 ml of RIA buffer (0.05 M phosphate buffer pH 7.4, containing 0.i % HSA, 0 . 0 1 % NaN. and 0.i % Triton X-100). After the incubation for ° 48 hours at 4°C, 0.2 ~i of 66.0 % normal human plasma and 1.5 ml of 22.0 polyethylen glycohol (#6000, Kanto Chemical Co., Tokyo, Japan) were added, vortexed and centrifuged for 15 minutes at 4°C. The supernatant was aspirated and the precipitate was counted by a -,-counter. Recovery of standard CRF added to plasma by this extraction method was 85.6 _+ 3.4 % for the mean concentration of 20 pg/ml. Sensitivity of the CRF assay was 4 pg/tube and intra and inter-assay coefficiency of variation of the CRF-RIA were 3.6 and 14.3 %, respectively. The antiserum used for extraction, recognized 24 to 41 amino acid sequences of hCRF. For CRF RIA, an antiserum which specifically recognizes the C-terminal amino acid sequence of hCRF was used. Cross-reactivity of the antiserum employed for RIA was, on a molar basis, 100% with (24-41)hCRF and (27-41)hCRF and 70% with (21, 38)Met(O)hCRF, but less than 0.1% with (I-20)hCRF, (l-41)ovine CRF, (l-44)h-growth hormone-releasing factor (GRF), (I-39)hACTH, hB-lipotropin (LPH), h%LPH, hS-endorphin, h-corticotropin-like intermediate lobe peptide (CLIP), ~-melanccyte-stimulating hormone (MSH), porcine BMSH, thyrotropin-releasing hormone (TRH), and luteinizing hormonereleasing hormone (LH-RH). Tyr(0)hCRF (Peptide Inst. Osaka, Japan) was iodinated with 125-INa by the chloramine T method (9) and used as a tracer after purification with the immunoaffinity column described above. Synthetic (I-41)hCRF (Peptide Inst., Osaka, Japan) was used as reference standard. 140 ml of pooled normal human plasma was extracted with ~ o a f f i n i t y chromatography described above, lyophilized, reconstituted in RIA buffer and applied to a Sephadex G-50 column (0.9 x 28 cm) and eluted with RIA buffer. 0.46 ml fraction was collected and CRF in each fraction was assayed with CRF-RIA. ACTH and cortisol were determined by RIA's, respectively, as described previously (10). Statistical significance was examined using Student's paired t-test, and results were presented as mean _+ S.E. unless otherwise stated. Results
Vol. 40, No. 17, 1987
Diurnal Rhythm in Plasma CRF in Man
10
*
**
A
1653
13<0.05
p
0
25
0 20
RR n
v
8
/nk
;S I. 0
0
120O 1800 Clock time
FIG. i. Plasma immunoreactive CRF (top), ~ (middle) and cortisol (bottom) concentrations at various time of the day in normal subjects (n=6). Asterisks indicate significant differences from corresponding values at 0600h. * p<0.05, ** p<0.001. Most of IR-CRF (84.1% of total) was eluted in a position of synthetic hCRF, although small portion (15.9 % of total) of IR-CRF conloonent was eluted between the synthetic CRF and total volume of the collins. The mean plasma CRF concentrations in six normal subjects at various times of the day are shown Fig. i. A statistically significant difference was observed between these concentrations, with the highest level (7.0 _+ 1.2 pg/ml) at 0600h. The lowest CRF level (1.7 -+ 0.2 pg/ml) was observed at 1800h which was not statistically different from that (1.9 -+ 0.4 pg/ml) obtained at 2200h. Changes in plasma concentrations of ACTH and cortisol simultaneously c~termined, are also shown in Fig. i. As expected, clear diurnal variations were demonstrated with the heighest value at 0600h (44.6 _+ 8.1 pg/ml and 15.9 + 2.0 ug/dl, respectively) and with the lowest at 2200h (12.3 _+ 2.8 pg/ml and 4.6 -+ 1.0 ug/dl, respectively). Diurnal changes in these three hormones appeared to parallel each other. The highest values were observed in the early morning (at 0600h) and significantly lower values in the evening (at 1700h and 2200h) for each of the hormones studied. D~ion Gel filtration analysis of the plasma extract indicated that most of IR-CRF measured in the present study was chromatographically indistinguishable from synthetic hCRF. Smaller portion of IR-CRF eluted between the synthetic CRF and total volume of the Sephac~x G-50 column may represent fragment(s) of hCRF or immunologically closely related material(s) to hCRF.
1654
Diurnal Rhythm in Plasma CRF in Man
Vol. 40, No. 17, 1987
In the present study, a clear diurnal rhythm was confirmed in plasma levels of both ACTH and cortisol as in the previous studies (i, ii, 12). Significant diurnal variation in plasma levels of CRF was also demonstrated in this study, which appeared to parallel those of ACTH and cortisol. Plasma concentrations of all of the hormones were significantly higher in the early morning and lower in the early to late evening. CRF has been shown to stimulate ACTH secretion from the pituitary by acting directly on the pituitary corticotrophs (5), and in patients with corticotroph-sparing secondary adrenal insufficiency, Avgerinos et al. have shown that CRF, when administered in an appropriate manner, is able to restore the circadian rhythm of ACTH and cortisol (13). Thus, our results strongly suggest that the diurnal variation in plasma ACTH and cortisol are regulated, at least in part, by a similar diurnal variation in CRF secretion in normal subjects. However, the persistent circadian rhythm of ACTH has been demonstrated, despite a constant plasma CRF level during the synthetic CRF infusion (14). Thus, it remains to be established whether the other factors, such as vasopressin (15, 16, 17), oxytocin (15), catecholamines (15, 17) and angiotensin II (18), which are known to modulate or stimulate ACTH secretion, might contribute to diurnal variation in ACTH and cortisol secretion. ~:knowledgements We are grateful to Miss Yukiko Hayashi and Miss Harue Sekigaml for their excellent technical assistance. We also thank The NIAMDD and The National Pituitary Agency for generously supplying RIA reagents for the ACTH assay. This work was supported in part by a research grant for intractable disease from the Japanese Ministry of Health and Welfare, by a research grant from the Japanese Ministry of Education, Science and Culture and by a research grant from Sankyo Foundation of Life Science. References i. K. TANAKA, W.E. NICHOLSON and D.N. ORTH, J. Clin. Endcrinol. Metab. 46 883-890 (1978) 2. P.E. MULLEN, W.J. JEFFCOATE, C. LINSELT., R. HOWARD and L.H. RESS, Clin. Endocrinol. ii 533-539 (1979) 3. R.R.M. DH~qT, C. GUIT.T.F24_INAULT,L.H. ALBERT, B.I. POSNER, B.M. COX and A. GOLDSTEIN, J. Clin. Endocrinol. Metab. 52 942-947 (1981) 4. S. SHIBAHARA, Y. MORIMOTO, Y. FURUTANI, M. NOTAKE, H. TAKAHASHI, S. SHIMIZU, S. HORIKAWA and S. NUMA, ~MBO J. 2 775-779 (1983) 5. W. VALE, J. SPIESS, C. RIVIER and J. RIVIER, Science 213 1394-1397 (1981) 6. T. SUDA, N. TOMORI, F. TOGAWA, T. MOURI, H. DEMURA and K. SHIZUME, J. Clin. Endocrinol. Metab. 59 861-866 (1984) 7. D.M. GIBBS and W. VALE, Endocrinolgy iii 1418-1420 (1982) 8. S.C. MARCH, I. PARIKH and P. CUATRECASAS, Anal. Biochem. 60 149-152 (1974) 9. D.N. ORTH, K. TANAKA and W.E. NICHOLSON, Methods of Hormone Radioimmunoassay, p235-313, Academic Press, New York (1971) i0. T. WATABE, K. TANAKA, M. HASEGAWA, S. MIYABE and N. SHIMIZU, Endocrinol. Japon. 32 771-779 (1985) ii. D.N. ORTH, D.P. Island and G.W. LIDDLE, J. Clin. Endocr. 27 549-555 (1967) 12. E.D. WEIZMAN, D. FUKUSHIMA, C. NOGEIRE, H. ROFFWARG, T.F. GALLAGHER and L. HEI.T.MAN J. Clin. Endocr. 33 14-22 (1971) 13. P.G. AVGERINOS, T.H.SCH0]hMEYER~ P.W. GOLD, T.P. TOMAI, D.L. LORIAUX, R.J. SHERINS, G.B. CUTLER and G.P. CPLROUSOS, J. Clin. Endocrinol. Metab. 62 816-821 (1986) 14. H.M. SCHULTE, G.P. CHROUSOS, P.W. GOLD, J.D. BOOTH, E.H. OLDFIELS, G.B. CUTLER and D.L. LORIAUX, J. Clin. Invest. 75 1781-1785 (1985)
Vol. 40, No. 17, 1987
Diurnal Rhythm in Plasma CRF in Man
15. W. VALE, J. VAUGHAN, M. SMITH, G. YAMAMOTO, J. R I V I E R a n d C. RIVIER, Endocrinology 113 1121-1131 (1983) 16. S.W.J. LAMBERTS, T. VERLEUN, R. OOSTEROM, F. J O N G a n d W.H.L. HACKENG, J. Clin. Endoclinol. Metab. 58 298-303 (1984) 17. C. RIVIER and W. VALE, Nature 305 325-327 (1983) 18. E. SPINEDI and A. NEGRO-VILAR, 5ke.uroendocrinology 37 446-453 (1983)
1655
Pergamon Journals
DIURNAL RHYTHM OF PLASMA IM~4UNOREACTIVE CORTICOTRf~IN-N~.FASING FACTOR IN NORMAL SUBJECTS Toshio Watabe*, Koshi Tanaka**, M~nehito Kumagae*, Sachiko Itoh*, Mitsutoshi Hasegawa**, Toshiyuki Horiuchi**, Syuji Miyabe**, HidetoOhno*** and Naokata Shimizu* Third Department of Medicine*, First Department of Medicine**, Teikyo University School of Medicine, 2-11-1, Kaga, Itabashi-ku, Tokyo 173, Japan and Central Research Laboratory***, Mitsubish Petrochemical Co. Ltd., 1315, Ami-machi, Inashiki-gun, Ibaraki-ken, 300-03, Japan. (Received in final form February 2, 1987)
summary Plasma corticotropin-releasing factor (CRF), corticotropin (ACTH) and cortisol levels were simultaneously determined by radioimmunoassays at 0600h, 1200h, 1800h and 2200h in six normal subjects, in order to examine whether the diurnal rhythm in plasma CRF exists and how it correlates to the diurnal rhythm in plasma ACTH and cortisol concentration. The highest CRF level was observed at 0600h (7.0 ± 1.2 pg/ml) and significantly lower levels (p<0.01) at 1800h (1.7 ± 0.2 pg/ml) and 2200h (1.9 ± 0.4 pg/ml). A clear diurnal rhythm was demonstrated in plasma ACTH and cortisol levels, with the highest values at 0600h (44.6 ± 8.1 pg/ml and 15.9 ± 2.0 ~g/dl, respectively) and the lowest at 2200h (12.3 ± 2.8 pg/ml and 4.6 ± 1.0 ~g/ml, respectively). These results suggest that the diurnal rhythm in ACTH and cortisol is under the regulation, at least in part, of the diurnal rhythm in CRF secretion. It has been well documented that there exists a diurnal rhythm in secretion of adrenocorticotropic hormone (ACTH) (i, 2) as well as other proopiomelanocortin (POMC)-relatedpeptides, such as B-lipotropin (i, 2) and B-endorphin (3). The release of ACTH and POMC-relatedpeptides from the pituitary, including diurnal variation, is assumed to be controlled mainly by corticotropin-releasing factor (CRF), although diurnal rhythm in CRF secretion in man has not been proven as yet. The amino acid sequence of human CRF (hCRF) was deduced recently from the nucleotide sequence of cloned DNA (4), following the isolation of CRF from ovine hypothalami by Vale et. al. (5). The availability of synthetic replicate of hCRF has made it possible to measure hCRF by radioimmunoassay (RIA) in various tissues (6) and in plasma (7). In the present study, imm~noreactive CRF (IR-CRF) was measured in plasma of normal subjects at various times of the day to examine whether there is a circadian variation in the plasma CRF concentration and how it correlates to those in plasma ACTH and cortisol.
0024-3205/87 53.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
1652
Diurnal Rhythm in Plasma CRF in Man
Vol. 40, No. 17, 1987
Materials and M~thods Six normal male subjects were studied after informed consent was obtained from each subject. They were aged from 23 to 31 (average 26.0) years old a n d w e i g h e d from 55 to 70 (average 62.2) kg. The subjects participated in this study while maintaining their customary daily routines of activity and meals throughout the study period. Blood specimens were obtained at 0600h, 1200h, 1800h and 2200h from an antecubital vein into a tube containing EDTA3K. Plasma was separated at 4°C by centrifugation and stored at -20°C until assay. Plasma IR-CRF was measured by a specific RIA after extraction of 20 ml plasma with immunoaffinity chromatography. CRF antiserum was partially purified by precipitation of -~-globulin with 50 % saturated ammonium sulfate and dialyzed. The 3"-globulin fraction was further purified with DEAE cellulose column, lyophilized and coupled to activated CH Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden) as reported (8). A portion (2.0 ml) of the antibody-coupled Sepharose 4B was packed in a small column and 20 ml of sample plasma was applied. The column was washed with 3.0 ml of 0.05 M phosphate buffer pH 7.4, containing 0.45 % NaCI and 0.i % Tween 80, followed by 3.0 ml of distilled water. The retained CRF was eluted with 2.5 ml of IN acetic acid, lyophilized and reconstitated with 0.7 ml of RIA buffer just prior to assay. Synthetic CRF standards or r e c o n s t i t u t ~ s a m p l e s were incubated with antiserum (final dilution 1:1,000,000) and I--D-CRF in a total volume of 0.5 ml of RIA buffer (0.05 M phosphate buffer pH 7.4, containing 0.i % HSA, 0 . 0 1 % NaN. and 0.i % Triton X-100). After the incubation for ° 48 hours at 4°C, 0.2 ~i of 66.0 % normal human plasma and 1.5 ml of 22.0 polyethylen glycohol (#6000, Kanto Chemical Co., Tokyo, Japan) were added, vortexed and centrifuged for 15 minutes at 4°C. The supernatant was aspirated and the precipitate was counted by a -,-counter. Recovery of standard CRF added to plasma by this extraction method was 85.6 _+ 3.4 % for the mean concentration of 20 pg/ml. Sensitivity of the CRF assay was 4 pg/tube and intra and inter-assay coefficiency of variation of the CRF-RIA were 3.6 and 14.3 %, respectively. The antiserum used for extraction, recognized 24 to 41 amino acid sequences of hCRF. For CRF RIA, an antiserum which specifically recognizes the C-terminal amino acid sequence of hCRF was used. Cross-reactivity of the antiserum employed for RIA was, on a molar basis, 100% with (24-41)hCRF and (27-41)hCRF and 70% with (21, 38)Met(O)hCRF, but less than 0.1% with (I-20)hCRF, (l-41)ovine CRF, (l-44)h-growth hormone-releasing factor (GRF), (I-39)hACTH, hB-lipotropin (LPH), h%LPH, hS-endorphin, h-corticotropin-like intermediate lobe peptide (CLIP), ~-melanccyte-stimulating hormone (MSH), porcine BMSH, thyrotropin-releasing hormone (TRH), and luteinizing hormonereleasing hormone (LH-RH). Tyr(0)hCRF (Peptide Inst. Osaka, Japan) was iodinated with 125-INa by the chloramine T method (9) and used as a tracer after purification with the immunoaffinity column described above. Synthetic (I-41)hCRF (Peptide Inst., Osaka, Japan) was used as reference standard. 140 ml of pooled normal human plasma was extracted with ~ o a f f i n i t y chromatography described above, lyophilized, reconstituted in RIA buffer and applied to a Sephadex G-50 column (0.9 x 28 cm) and eluted with RIA buffer. 0.46 ml fraction was collected and CRF in each fraction was assayed with CRF-RIA. ACTH and cortisol were determined by RIA's, respectively, as described previously (10). Statistical significance was examined using Student's paired t-test, and results were presented as mean _+ S.E. unless otherwise stated. Results
Vol. 40, No. 17, 1987
Diurnal Rhythm in Plasma CRF in Man
10
*
**
A
1653
13<0.05
p
0
25
0 20
RR n
v
8
/nk
;S I. 0
0
120O 1800 Clock time
FIG. i. Plasma immunoreactive CRF (top), ~ (middle) and cortisol (bottom) concentrations at various time of the day in normal subjects (n=6). Asterisks indicate significant differences from corresponding values at 0600h. * p<0.05, ** p<0.001. Most of IR-CRF (84.1% of total) was eluted in a position of synthetic hCRF, although small portion (15.9 % of total) of IR-CRF conloonent was eluted between the synthetic CRF and total volume of the collins. The mean plasma CRF concentrations in six normal subjects at various times of the day are shown Fig. i. A statistically significant difference was observed between these concentrations, with the highest level (7.0 _+ 1.2 pg/ml) at 0600h. The lowest CRF level (1.7 -+ 0.2 pg/ml) was observed at 1800h which was not statistically different from that (1.9 -+ 0.4 pg/ml) obtained at 2200h. Changes in plasma concentrations of ACTH and cortisol simultaneously c~termined, are also shown in Fig. i. As expected, clear diurnal variations were demonstrated with the heighest value at 0600h (44.6 _+ 8.1 pg/ml and 15.9 + 2.0 ug/dl, respectively) and with the lowest at 2200h (12.3 _+ 2.8 pg/ml and 4.6 -+ 1.0 ug/dl, respectively). Diurnal changes in these three hormones appeared to parallel each other. The highest values were observed in the early morning (at 0600h) and significantly lower values in the evening (at 1700h and 2200h) for each of the hormones studied. D~ion Gel filtration analysis of the plasma extract indicated that most of IR-CRF measured in the present study was chromatographically indistinguishable from synthetic hCRF. Smaller portion of IR-CRF eluted between the synthetic CRF and total volume of the Sephac~x G-50 column may represent fragment(s) of hCRF or immunologically closely related material(s) to hCRF.
1654
Diurnal Rhythm in Plasma CRF in Man
Vol. 40, No. 17, 1987
In the present study, a clear diurnal rhythm was confirmed in plasma levels of both ACTH and cortisol as in the previous studies (i, ii, 12). Significant diurnal variation in plasma levels of CRF was also demonstrated in this study, which appeared to parallel those of ACTH and cortisol. Plasma concentrations of all of the hormones were significantly higher in the early morning and lower in the early to late evening. CRF has been shown to stimulate ACTH secretion from the pituitary by acting directly on the pituitary corticotrophs (5), and in patients with corticotroph-sparing secondary adrenal insufficiency, Avgerinos et al. have shown that CRF, when administered in an appropriate manner, is able to restore the circadian rhythm of ACTH and cortisol (13). Thus, our results strongly suggest that the diurnal variation in plasma ACTH and cortisol are regulated, at least in part, by a similar diurnal variation in CRF secretion in normal subjects. However, the persistent circadian rhythm of ACTH has been demonstrated, despite a constant plasma CRF level during the synthetic CRF infusion (14). Thus, it remains to be established whether the other factors, such as vasopressin (15, 16, 17), oxytocin (15), catecholamines (15, 17) and angiotensin II (18), which are known to modulate or stimulate ACTH secretion, might contribute to diurnal variation in ACTH and cortisol secretion. ~:knowledgements We are grateful to Miss Yukiko Hayashi and Miss Harue Sekigaml for their excellent technical assistance. We also thank The NIAMDD and The National Pituitary Agency for generously supplying RIA reagents for the ACTH assay. This work was supported in part by a research grant for intractable disease from the Japanese Ministry of Health and Welfare, by a research grant from the Japanese Ministry of Education, Science and Culture and by a research grant from Sankyo Foundation of Life Science. References i. K. TANAKA, W.E. NICHOLSON and D.N. ORTH, J. Clin. Endcrinol. Metab. 46 883-890 (1978) 2. P.E. MULLEN, W.J. JEFFCOATE, C. LINSELT., R. HOWARD and L.H. RESS, Clin. Endocrinol. ii 533-539 (1979) 3. R.R.M. DH~qT, C. GUIT.T.F24_INAULT,L.H. ALBERT, B.I. POSNER, B.M. COX and A. GOLDSTEIN, J. Clin. Endocrinol. Metab. 52 942-947 (1981) 4. S. SHIBAHARA, Y. MORIMOTO, Y. FURUTANI, M. NOTAKE, H. TAKAHASHI, S. SHIMIZU, S. HORIKAWA and S. NUMA, ~MBO J. 2 775-779 (1983) 5. W. VALE, J. SPIESS, C. RIVIER and J. RIVIER, Science 213 1394-1397 (1981) 6. T. SUDA, N. TOMORI, F. TOGAWA, T. MOURI, H. DEMURA and K. SHIZUME, J. Clin. Endocrinol. Metab. 59 861-866 (1984) 7. D.M. GIBBS and W. VALE, Endocrinolgy iii 1418-1420 (1982) 8. S.C. MARCH, I. PARIKH and P. CUATRECASAS, Anal. Biochem. 60 149-152 (1974) 9. D.N. ORTH, K. TANAKA and W.E. NICHOLSON, Methods of Hormone Radioimmunoassay, p235-313, Academic Press, New York (1971) i0. T. WATABE, K. TANAKA, M. HASEGAWA, S. MIYABE and N. SHIMIZU, Endocrinol. Japon. 32 771-779 (1985) ii. D.N. ORTH, D.P. Island and G.W. LIDDLE, J. Clin. Endocr. 27 549-555 (1967) 12. E.D. WEIZMAN, D. FUKUSHIMA, C. NOGEIRE, H. ROFFWARG, T.F. GALLAGHER and L. HEI.T.MAN J. Clin. Endocr. 33 14-22 (1971) 13. P.G. AVGERINOS, T.H.SCH0]hMEYER~ P.W. GOLD, T.P. TOMAI, D.L. LORIAUX, R.J. SHERINS, G.B. CUTLER and G.P. CPLROUSOS, J. Clin. Endocrinol. Metab. 62 816-821 (1986) 14. H.M. SCHULTE, G.P. CHROUSOS, P.W. GOLD, J.D. BOOTH, E.H. OLDFIELS, G.B. CUTLER and D.L. LORIAUX, J. Clin. Invest. 75 1781-1785 (1985)
Vol. 40, No. 17, 1987
Diurnal Rhythm in Plasma CRF in Man
15. W. VALE, J. VAUGHAN, M. SMITH, G. YAMAMOTO, J. R I V I E R a n d C. RIVIER, Endocrinology 113 1121-1131 (1983) 16. S.W.J. LAMBERTS, T. VERLEUN, R. OOSTEROM, F. J O N G a n d W.H.L. HACKENG, J. Clin. Endoclinol. Metab. 58 298-303 (1984) 17. C. RIVIER and W. VALE, Nature 305 325-327 (1983) 18. E. SPINEDI and A. NEGRO-VILAR, 5ke.uroendocrinology 37 446-453 (1983)
1655