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Diurnal rhythm in total serum thyroxine levels
Diurnal Rhythm in Total Serum Thyroxine Levels B y PHILIPPE DE COSTRE, ULRICH BUHLER, LESLIE J. DEGROOT,
AND SAMUEL REFETOFF The diurnal variation of serum thyroxine, plasma proteins, and hematocrit were studied in six patients (euthyroid, hypothyroid, and thyrotoxie) and in two obese subjects during total fast. Variations of 7% to 34% were observed in serum total protein, hematocrit, proteinbound iodine, totul thyroxine, thyroxine binding globulin, and prealbumin binding capacity, and thyroxine dialyzable fraction, but not in the concentration of serum free thyroxine. Similar and parallel diurnal variation was observed during studies of the disappearance of l~lIlabeled thyroxine and 125I-labeled albumin. The maximal daily concentration occurred between 10:00 a.m. and 2:00 p.m., and the minimum occurred around 2:00 a.m. These diurnal variations were
not related to food intake or adrenocortical function but were influenced by posture. A reversed cyclic change could be produced by reversing the normal sleep-waking pattern. Because of reciprocal changes in the concentration of total serum thyroxine and thyroxine dialyzable fraction, the serum-free thyroxine concentration remained constant. These rhythmic changes are apparently caused by m o v e m e n t of fluid into and out of the vascular compartment. This factor may be an important cause of scatter in the observations recorded during studies of slowly diffusible plasma constituents if repeated blood sampling is nut performed under the same conditions.
R
ENBOURN, IN 1947, DESCRIBED RHYTHMS for hemoglobin, hematocrit, and plasma proteins characterized by a daily minimum level for these parameters around 2:30 a.m. He concluded that, although the rhythms might be influenced by posture, sleep, exertion, or food intake, these changes were not directly due to any of these factors. 1 A similar nocturnal depression of 13~I-labeled thyroxine (Ta-~31I) concentration was noted by Walfish et al., who obtained frequent blood samples during a study of T4-13~I disappearance from plasma/ The concentration of T~-~3aI in 2:00 p.m., 8:00 p.m., and 2:00 a.m. samples was significantly lower than the theoretical values corresponding to the best fit of a regression line drawn through the values of the 8:00 a.m. samples. From the Thyroid Study Unit, Department of Medicine, University of Chicago, Chicago, Ill., and the Clinical Research Center, Massachusetts Institute of Technology, Cambridge, Mass. Received for publication February 11, 1971. Supported in part by USPHS Grants A M 13377 and A M 13643, by American Cancer Society Grant P-298 E, and by the John A. Hartford Foundation, New York. PHILIPPE DE COSTRE,M.D.: Research Fellow, Clinical Research Center, Massachusetts Institute of Technology, Cambridge, Mass. ULRICHBUHLER,M.D. : Research Fellow, Clinical Research Center, Massachusetts Institute of Technology, Cambridge, Mass. LESLIE J. DEGRooT, M.D.: Professor of Medicine and Head, Thyroid Study Unit, University of Chicago Pritzker School of Medicine, Chicago, Ill. SAMUELREFETOFF, M.D.: Assistant Professor of Medicine, University of Chicago Pritzker School of Medicine, Chicago, Ill.
782
METABOLISM, VOL. 20, No. 8 (AuGuST), 1971
D~RNAL RHYTHM
783
O n first evaluation, this d i u r n a l r h y t h m i n thyroxine (T4) d i s a p p e a r a n c e rate suggested that there were cyclic fluctuations in the utilization of this h o r m o n e , b u t other data suggested that the variations m a y be due to aonspecific factors such as p l a s m a v o l u m e fluctuation. We have investigated the effect of changes in feeding schedule, sleep-waking cycle, upright or r e c u m b e n t posture, a n d a d r e n a l f u n c t i o n o n T4 c o n c e n t r a t i o n in serum. O u r data agree with the findings of Renbour,n a n d of Walfish et al. i n the general description of the fluctuations b u t indicate that the variation in total serum T4 c o n c e n t r a t i o n is related to changes in the patient's posture. T h e data indicate the i m p o r t a n c e of p l a s m a v o l u m e alteration in d e t e r m i n a t i o n of d i u r n a l rhythms, particularly for substances circulating b o u n d to proteins. O u r observations emphasize the influence of p l a s m a dilution or c o n c e n t r a t i o n o n e v a l u a t i o n of the rate of disappearance of T4 f r o m p l a s m a a n d suggest a m e t h o d for correcting the scatter of individual values w h e n b l o o d sampling is n o t p e r f o r m e d u n d e r exactly similar conditions. MATERIALS AND METHODS One euthyroid normal subject ( J . J . ) , o n e patient with euthyroid Graves' disease (L.M.), two patients with moderate hyperthyroidism due to Graves' disease (A.D. and A.W.), and two moderately hypothyroid patients (J.N. and C.B.) were studied. They received a simultaneous intravenous injection of 40 #Ci of T4-1zlI and 10/zCi of 125I-labeled human serum albumin (12gI-RISA or Albumin-12gI). Thyroidal iodide recirculation was blocked in one patient (JJ.) by administration of potassium iodide, 100 mg daily. The protein bound lzlI (PBlaaI) and pB125I were measured 10 min, 30 rain, 60 min, 120 rain, 8 hr, and 24 hr after the injection, and then twice daily. After a uniform disappearance slope was attained for both PB13~I and PBlZSI, diurnal variations were studied by repeated sampling of blood at 8 a.m., 10 a.m., 1 p.m., 5 p.m., 9 p.m., and 2 a.m. During the period of frequent sampling, the PBlZ7I, hematocrit, and total plasma proteins (TP) were measured (using a T.C. Refractometer, American Optical Corporation, Buffalo, N. Y.) in addition to isotope labeled trichloroacetic acid (TCA) precipitable iodine. Under conditions of this study, determinations of PB131I values on replicate samples gave a standard deviation of less than 5% of the mean. pBleTI values were determined by Boston Medical Laboratory. The usual standard deviation on replicate assays of one sample in their laboratory is also under 5% of the mean. The maximal T 4 binding capacity of thyroxine binding globulin (TBG) and thyroxine binding prealbumin (TBPA),3 concentration of plasma free fatty acids (FFA),4 tyrosine,5 total thyroxine (TT4),6 and the T 4 dialyzable fraction (T4-DF)7 were also evaluated in some patients. Changes in 131I and 1251 activity in thyroid and liver were assessed by external counting over these organs using a Nuclear Chicago Scintillation Gamma Ray Counter. In the initial period patients were kept on a normal sleepwaking cycle (sleeping between 10:00 p.m. and 8:00 a.m.) and on a regular diet with meals at 8:00 a.m., noon, and 6:00 p.m. After completion of studies for a normal schedule, the patients were studied in various experimental conditions. Each subject served as his own control. In two subjects (J.N. and J.J.), meals were given as equal aliquots of food every 6 hr, while the patients were recumbent from 10:00 p.m. to 8:00 a.m. as usual; the 2:00 a.m. meal was consumed while the subject remained recumbent. In three subjects (J.L, C.B., and J.N.) the sleep-waking cycle was reversed. When awake patients were required to be up and about. Meals were scheduled at the usual time, but lunch was omitted. In three subjects (J.N., C.B,, and L.N.) the 17-hydroxy-corticosteroid and 17-ketosteroid excretion in the urines was studied during a normal sleep-waking cycle. Dexamethasone, .5 mg every 6 hr, was then given for 3 days while the patients were maintained on the usual sleep-waking cycle and the studies were repeated. The T4-125I plasma disappearance rate was evaluated in two obese patients (G.S. and
784
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Fig. 1.--Typical study showing diurnal fluctuation of T4AslI and Albumin-12~I, hematocrit, total protein, and PB12q. N o diurnal fluctuation is apparent in thyroid and liver isotope content.
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S,M.) during total fasting after injection of T4-131I and Albumin-125I. PBlalI, PB125I, sermn total proteins, thyroxine and dialyzable thyroxine fraction were measured in each sample, and the free T 4 concentration calculated. The slopes of the curves and the intercepts were evaluated by means of the method of least squares. RESULTS
In the normal pattern, diurnal variations occurred in all patients and were similar in direction. For this reason subsequent observations are presented without reference to thyroid diagnosis. Data for one period of frequent sampling (patient A.W.) is detailed in Fig. 1. The curves are given for a period of more than 24 hr. No consistent change is observed in thyroid and liver radioactivity, while the other parameters, except for tyrosine, show a similar modification. The maximal concentration of the various constituents in plasma occurred between 8:00 a.m. and 1:00 p.m., and the minimum level is observed around 2:00 a.m. The tyrosJne concentration obtained in two patients (A.W. and A.O.) and the free fatty acids in one patient (A.O.) did not follow the same rhythm. The PBI~II curves obtained for each patient during a normal sleep cycle are given in Fig. 2. The values are expressed as a percentage of the 2:00 a.m. values. The seven curves show the same general shape and their mean value is
DIURNAL RHYTHM
Fig. 2.--Diurnal fluctuations of plasma T4-131I for seven different periods in six patients. Concentrations are expressed as per cent of 2:00 a.m. value on second day taken as 100%. Thin lines: individual patient's data. Thick line: mean for all observations. The 11% difference between 2:00 a.m. values on two consecutive days is due to T4 degradation. Mean fractional degradation for all patients is 10.6%/day.
785
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characterized by a maximal PB~II level between 10:00 a.m. and 1:00 p.m., and a minimal concentration at 2:00 a.m. There is on the average a 19% difference between the maximum value at 10:00 a.m. and the minimum at 2:00 a.m. The mean plasma T4-1a31 disappearance slope is .106 fraction per day for the six patients. On average the biologic decay of thyroxine 1~1I would account for 16 h r / 2 4 hr )< 0.11 fr./day X 19% = 8% of this decline. Thus the decrease in PBIZlI levels from 12:00 noon to 2:00 a.m., attributed to the diurnal rhythm, is on average 11% of the noon value. A study of the reversal of the sleep-waking cycle was performed in four patients; a typical result is shown in Fig. 3. It is evident that the usual pattern is demonstrated for the normal sleep-waking cycle. As soon as the patient's sleep cycle is reversed, there is a reversal in the rhythm of the TP, T4-1alI and AlbuminJ25I concentrations in plasma. The minimal concentratio.n, at 2:00 a.m. for the normal cycle, is altered to a minimal concentration at 2:00 p.m. during the reversed sleep schedule. The minimal concentration corresponds i.n each case to the recumbent position. The influence of changing the feeding cycle was studied in three patients given four equal feedings at 6-hr intervals. The diurnal rhythm minimum was at 2:00 a.m. there was ,no change in the shape of the curve. The influence of the adrenal steroid rhythm was analyzed in three patients; a typical study is presented in Fig. 4. The urinary 17-hydroxycorticoid excretion demonstrated the usual diurnal variation during the sleeping and the waking period. After suppression of adrenal function by 2 m g / d a y of dexamethasone, there was no change in the rhythm for total protein, PB"~[, or AlbuminYSI in plasma. The practical importance of postural changes in the evaluation of T4 and
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Fig. 3.mInfluence of posture on plasma concentrations of T4-131I, Albumin-125I, and total proteins. Thick line: periods of recumbency. Reverse sleep cycle values (circles) for Albumin-125I and T~-131I are at higher level than normal sleep cycle values (triangles) because of gradual biologic decay of labeled compounds. Daytime sleep pattern reverses usual diurnal cycle and causes low values at noon. albumin metabolism is demonstrated in Fig. 5. This obese subject was on a total fast for 1 wk prior to and during the study. During the 12 days of this study, blood was taken each morning before the patient got up, except on days 6 and 10. On these days there was a distinct increase in plasma protein concentration. The slopes of the T4-13lI and Albumin-125I disappearance curves, plotted by the method of least squares, for this data are given in lines A and A', Fig. 5. When the albumin-125I and T4-a3H values are related to the protein concentration, deviations from the straight line become ne~igible, and the slopes have slightly different values (lines B and B', Fig. 5). Differences of 7% and 33%, respectively, were found for T4 and albumin slopes, aaad differences of 4% and 17%, respectively, for T4 and albumin distribution spaces. TBG and TBPA capacity, T4, and dialyzable fraction were determined on samples obtained from the same patient. It may be seen that absolute flee T4 remained relatively constant despite significant variation of total T4 concentration (Table 1), and
DIURNAL RHYTHM
787
that variations in total T4, TBG, and TBPA were directly proportional, and the T4 dialyzable fraction inversely proportional to the total serum protein concentration. Similar changes were observed in a thyrotoxic patient (A.O.) during one complete diurnal cycle (Table 2). DISCUSSION When frequent blood samples are analyzed during a study of the disappearance of labeled T4 from plasma, a daily maximum plasma concentration of radioactive PBI is observed between 10:00 a.m. and 2:00 p.m., while a miniI
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Fig. 4.--Influence of adrenocortical secretion on plasma concentration. Left panel represents normal pattern with normal diurnal variations of 17-hydroxycorticoid excretion. Right panel shows maintenance of rhythm after suppression of adrenocortical secretion with dexamethasome. Four days elapsed between first and second studies. Dexamethasone does not influence rhythm.
788
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Fig. 5 . - - I m p o r t a n c e of postural changes in evaluation of T4 and R1SA metabolism. Line A: data on T4-1311% dose/1; ten, related in line B to protein content of samples (% dose • 10 g T P ) . Line A ' : data on Albumin-levi % d o s e / l , related in line B' to protein content of samples (% dose • 10 g T P ) . Boxes identify corresponding half-times ( t 8 9 decay rates ( K ) , and distribution spaces (D.S.). m u m occurs a r o u n d 2 : 0 0 a.m. T h e la b e le d h o r m o n e c o n c e n t r a t i o n at 2 : 0 0 a.m. is on average 1 1 % b e l o w the 1 0 : 0 0 a.m. value. N o consistent reciprocal cha,nges were o b s e r v e d in liver T4 content, i n d i c a t i n g there was n o m o v e m e n t of h o r m o n e
Table L--Influence of Posture on Serum Total Proteins, T 4 Binding Proteins, and T4 Concentration D a y of Sampling
Position
4 recumbent 5 recumbent 6 standing 9 recumbent 10 standing % change from days 4 t o 6 % change from days9 to 10
Total Protein (g/100 ml)
Total T, Dialyzable Free Thyroxine Fraction Thyroxine (/Zg/100 ml) (%) (m/tg/100 ml)
0.056 0.0.57 0.047 0.052 0.040
3.36 3.28 3.29 3.07 3.16
T4 Binding Capacity TBG* TBPA (#g/100 ml)
6.2 6.2 7.3 6.3 7.8
6.0 5.7 7.0 5.9 7.9
21.4 20.2 24.5 21.5 25.4
• • • • •
0.9at 0.2 a 0.3 b 1.0a 0.9 b
116.2 102.8 132.0 120.2 146.7
+17.7
+16.7
-16.1
-2.1
+14.7
+13.6
+23.8
+33.9
-23.1
+2.9
+18.4
+22.1
* Mean _ standard deviation on four consecutive determinations of same sample. t Values not significantly different from each other are indicaed with same superscript. Those with different superscripts are significantly different at p<0.05 to p<0.001.
DIURNAL RHYTHM
789
Table 2 . - - D i u r n a l Variation of Thyroxine Concentration and Plasma Constituents in Patient A.O. With Thyrotuxicosis D u e to Graves' Disease Time of Day 7 : 30 10:00 5:00 2:00
a.m. a.m. p.m. a.m.
Total Protein (g/100 ml)
PB127I (#gI/100 ml)
6.4 7.1 7.2 6.3
13.2 14.4 15.2 13.0
T~ Dialyzable Free TI Binding Fraction Thyroxine Capacity of TBG Hematocrit (%) (m/xgTa/100 ml) (~tg/100 ml) (%) 0.097 -0.091 0.113
12.8 -13.8 14.7
16.3 20.0 21.1 16.2
43.0 48.5 44.5 40.0
in and out of this storage area. However, a change in this parameter might be too small to be detected by the external counting technique employed in our study. The cycle described for T4-131I (PBla~I) was paralled exactly by changes in Albumin-12~I, PB~27I, total thyroxine, total protein concentration, TBG and TBPA capacity, and hematocrit. Reversed cyclic changes were observed in the T4 dialyzable fraction, resulting in a stable free T4 concentration. The cyclic changes in serum T4 concentration were not influenced by the feeding rhythm nor by suppression of adrenal secretion. When the sleep-waking cycle was reversed, there was an immediate reversal of the rhythm with a minimum corresponding to the new sleeping period. This rhythm is similar in both degree of change and in periodicity to that described by Doe for plasma cortisone-binding globulinY Since Albumin-12~I leaves the intravascular space relatively slowly and is not accumulated in any organ, marked and cyclical changes in its concentration in blood are presumably good indications of plasma volume shifts. The existence of a rhythm for AlbuminJ2aI concentration, similar to that for T4, suggests that the changes observed are mostly due to diurnal variations in plasma volume, with reciprocal changes in plasma protein concentration. A redistribution of fluids in the body occurring with a change from the recumbent to the upright posture has long been recognized,TM and could explain the rhythm noted in these studies. Hydrostatic pressure in the lower part of the body is higher during upright standing than during recumbeny, and the equilibrium with the intravascular osmotic pressure is altered. According to this mechanism, upon upright standing there is a transfer of fluid to the extravascular space, which increases the concentration of nondiffusible plasma constituents until a new equilibrium is reached between two pressures. This phenomenon could explain the rhythm observed for hematocrit, PB~27I, and TT4 concentration, TBG capacity, and the relative constancy of the free T4 concentration. It is not necessary to hypothesize that T4 or TBG moved in and out of the vascular compartment, or that variations in T4 secretion or metabolism occurred. Cyclic hemodilution and hemoconcentration, by transfer of water, could explain the rhythm we have observed. The well-described diurnal cycle in urine flow is characterized by peak flow from 6:00 to 12:00 a.m., a decrease in the afternoon, and nadir between 12 midnight and 6:00 a.m21 This cycle is also reversed by a "reversed" sleep cycle. A similar rhythm is seen in patients kept recumbent throughout the 24-hr period32 This cycle could possibly play a causative role. in the cyclic variations of plasma protein concentration we have noted, although data are not sufficient to establish this relationship.
790
DE COSTRE ET AL~
It has been suggested1~ that muscular exercise increases osmotic pressure in muscle. Muscular work associated with upright posture could represent a second factor influencing transfer of fluids from intra- to extravascular compartments. The three mechanisms we have noted are believed to be sufficient to explain the rapid change in TT4 concentration when subjects get up in the morning, but do not adequately explain the tendency for TT4 concentration to resume a lower level in the afternoon. It is possible that other undetected regulatory mechanisms may be involved and may tend to buffer swings in concentration toward a "normal" value. Neither changes in the feeding rhythm nor suppression of adrenocortical secretion by dexamethasone were able to affect the T4 rhythm. This latter observation suggests that, at least for the short periods studied, the fluid transfers that we believe mediate the rhythm are not dependent upon cyclic variation in adrenal glucorticoid secretion. A diurnal rhythm has been reported for plasma amino acidsp ,~4 and concentration of tyrosine, phenylalanine ,and tryptophan being lower around 2:00 a.m. and higher around 10:00 a.m. For some amino acids the changes were 1.2 times the basal value, a level comparable to concentratio.n changes detected in our study. However, these amino acids are not bound tightly to protein, so this "rhythm" is presumably not caused by the same mechanism we postulate for the thyroxine rhythm. Evaluation of any metabolic rhythm must take into account the cyclic changes in the concentration of nondiffusible plasma constitutents, which could potentially interfere with the phenomenon studied. It is interesting to note that 11% change in the PBI can be related simply to the chronology of plasma sampling. The same conclusion is valid for other nondiffusible plasma constituents such as proteins or red blood cells. Modifications of that range may be completely independent of metabolic alterations. The induction of anesthesia by ether also results in a transient increase in plasma PBI concentration15 which may be related to changes in plasma volume. ~6 A last corollary of these observations is the importance of accurate standardization of the conditions of sampling for PBa27I and PBa3H determi~nations, and as a matter of fact, for any protein-bound compound in plasma. In the case of T~ disappearance rate evaluation, the scatter of the individual values, when other technical factors have been excluded, may be reduced by sampling under reproducible enviro,nmental conditions, or by correction of the radioactive PBI values for plasma protein concentration in the test sample. A reduction in the spread of the observations may lead to a greater accuracy of turnover data. The present investigation confirms the existence of a diurnal rhythm in the concentration of serum total thyroxine and certain other plasma constituents. The rhythm is related to a large extent to passive movement of fluids into and out of the vascular compartment. These diurnal variations are not related to food intake nor to adrenocortical function, but are influenced by the patient's posture. Diurnal variations in thyroid hormone secretion, a7 metabolism, or accumulation in liver may occur, but are not required to account for the fluctuations we have observed. The variations we observed in the total thyroxine concentration in serum are associated with constant concentration of free thyroxine, as would be expected from the known constancy of free thyroxine during dilution of
DIURNAL RHYTHM
791
plasma, is Since free thyroxine is considered to be the active h o r m o n a l form i n serum, capable of traversing cellular m e m b r a n e s a n d activating m e t a b o l i c processes, the d i u r n a l r h y t h m observed for s e r u m total thyroxine m a y n o t b e associated with a similar r h y t h m in intracellular h o r m o n a l c o n t e n t a n d degradation. Nevertheless, these r h y t h m s m a y b e a very i m p o r t a n t cause of scatter in the observations recorded during T4 t u r n o v e r studies if b l o o d s a m p l i n g is n o t perf o r m e d u n d e r the same conditions. A correction m e t h o d is p r o p o s e d to reduce the spread of observations a n d to increase the accuracy of the metabolic studies. ACKNOWLEDGMENT We are grateful to Mr. Richard J. Wurtman, Department of Nutrition and Food Sciences, Massachusetts Institute of Technology, Boston, Mass., for the determination of tyrosine, and to Dr. Philip Felig and Dr. George F. Cahill, Joslin Research Laboratory, Boston, Mass., for the determination of free fatty acid and permission to study the two obese patients during fast. REFERENCES 1. Renbourn, E. T.: Variation, diurnal and over longer periods of time, in blood haemoglobin, haematocrit, plasma protein, erythrocyte sedimentation rate, and blood chloride. J. Hygiene 45: 455, 1947. 2. Walfish, P. G., Britton, A., Melville, P. H., and Ezrin, C.: A diurnal pattern in the rate of disappearance of la~I-labeled 1-Thyroxine from the serum. J. Clin. Endocr. 21: 582, 1961. 3. Refetoff, S. and Selenkow, H. A.: Familial thyroxine-binding globulin deficiency in a patient with Turner's syndrome (XO) : genetic study of a kindred. New Eng. J. Med. 278: 1081, 1968. 4. Trout, D. L., Estes, E. H., Jr., and Friedberg, S. I.: Titration of free fatty acids of plasma: study of current methods and a new modification. J. Lipid Res. 1: 199, 1959. 5. Wurtman, R. J., Rose, C. M., Chou, C., and Larin, F. F.: Daily rhythms in the concentrations of various amino acids in human plasma. New Eng. J. Med. 279: 171, 1968. 6. Murphy, B. E. P., and Jachan, C.: The determination of thyroxine by competitive protein binding analysis employing an anion exchange resin and radiothyroxine. I. Lab. Clin. Med. 66: 161, 1965. 7. Fang, V. S., and Selenkow, H. A.: Determination of free thyroxine in serum by low-temperature equilibrium dialysis. Clin. Chem. 16: 185, 1970. 8. Reddy, W. J.: Modification of the Reddy-Jenkins-Thorn method for the esti-
mation of 17-hydroxycorticoids in urine. Metabolism 3: 489, 1954. 9. Doe, R. P., Fernandez, R., and Seal, U. S.: Measurement of corticosteroid-binding globulin in man. J. Clin. Endocr. 24: 1029, 1964. 10. Fawcett, I. K. and Wynn, V.: Effects of posture on plasma volume and some blood constituents. J. Clin. Path. 13: 304, 1960. 11. Smith, H.: The Kidney New York, Oxford University Press, 1951, p. 339. 12. Sirota, J. H., Baldwin, D. S., and Villarreal, H.: Diurnal variations of renal function in man. I. Clin. Invest. 29: 187, 1950. 13. Welt, L. J., Orloff, J., Kydd, D. M., and Oltman, J. E.: An example of cellular hyperosmolarity. I. Clin. Invest. 29: 935, t950. 14. Wurtman, R. J., Chou, C., and Rose, C. M.: Daily rhythm in tyrosine concentration in human plasma: Persistence on low protein diets. Science 158: 660, 1967. 15. Fore, W., Kohler, P., and Wynn, I.: Rapid redistribution of serum thyroxine during ether anesthesia. J. Clin. Endocr. 26: 821, 1966. 16. Albert, S. N.: Blood Volume. Springfield, II1., Thomas, 1963, p. 54. 17. Nicoloff, J. T.: A new method for the measurement of thyroid iodine release in man. J. Clin. Invest. 49: 1912, 1970. 18. Oppenheimer, J. H. and Surks, M. I.: Determination of free thyroxine in human serum: a theoretical and experimental analysis. J. Clin. Endocr., 24: 785, 1964.
AND SAMUEL REFETOFF The diurnal variation of serum thyroxine, plasma proteins, and hematocrit were studied in six patients (euthyroid, hypothyroid, and thyrotoxie) and in two obese subjects during total fast. Variations of 7% to 34% were observed in serum total protein, hematocrit, proteinbound iodine, totul thyroxine, thyroxine binding globulin, and prealbumin binding capacity, and thyroxine dialyzable fraction, but not in the concentration of serum free thyroxine. Similar and parallel diurnal variation was observed during studies of the disappearance of l~lIlabeled thyroxine and 125I-labeled albumin. The maximal daily concentration occurred between 10:00 a.m. and 2:00 p.m., and the minimum occurred around 2:00 a.m. These diurnal variations were
not related to food intake or adrenocortical function but were influenced by posture. A reversed cyclic change could be produced by reversing the normal sleep-waking pattern. Because of reciprocal changes in the concentration of total serum thyroxine and thyroxine dialyzable fraction, the serum-free thyroxine concentration remained constant. These rhythmic changes are apparently caused by m o v e m e n t of fluid into and out of the vascular compartment. This factor may be an important cause of scatter in the observations recorded during studies of slowly diffusible plasma constituents if repeated blood sampling is nut performed under the same conditions.
R
ENBOURN, IN 1947, DESCRIBED RHYTHMS for hemoglobin, hematocrit, and plasma proteins characterized by a daily minimum level for these parameters around 2:30 a.m. He concluded that, although the rhythms might be influenced by posture, sleep, exertion, or food intake, these changes were not directly due to any of these factors. 1 A similar nocturnal depression of 13~I-labeled thyroxine (Ta-~31I) concentration was noted by Walfish et al., who obtained frequent blood samples during a study of T4-13~I disappearance from plasma/ The concentration of T~-~3aI in 2:00 p.m., 8:00 p.m., and 2:00 a.m. samples was significantly lower than the theoretical values corresponding to the best fit of a regression line drawn through the values of the 8:00 a.m. samples. From the Thyroid Study Unit, Department of Medicine, University of Chicago, Chicago, Ill., and the Clinical Research Center, Massachusetts Institute of Technology, Cambridge, Mass. Received for publication February 11, 1971. Supported in part by USPHS Grants A M 13377 and A M 13643, by American Cancer Society Grant P-298 E, and by the John A. Hartford Foundation, New York. PHILIPPE DE COSTRE,M.D.: Research Fellow, Clinical Research Center, Massachusetts Institute of Technology, Cambridge, Mass. ULRICHBUHLER,M.D. : Research Fellow, Clinical Research Center, Massachusetts Institute of Technology, Cambridge, Mass. LESLIE J. DEGRooT, M.D.: Professor of Medicine and Head, Thyroid Study Unit, University of Chicago Pritzker School of Medicine, Chicago, Ill. SAMUELREFETOFF, M.D.: Assistant Professor of Medicine, University of Chicago Pritzker School of Medicine, Chicago, Ill.
782
METABOLISM, VOL. 20, No. 8 (AuGuST), 1971
D~RNAL RHYTHM
783
O n first evaluation, this d i u r n a l r h y t h m i n thyroxine (T4) d i s a p p e a r a n c e rate suggested that there were cyclic fluctuations in the utilization of this h o r m o n e , b u t other data suggested that the variations m a y be due to aonspecific factors such as p l a s m a v o l u m e fluctuation. We have investigated the effect of changes in feeding schedule, sleep-waking cycle, upright or r e c u m b e n t posture, a n d a d r e n a l f u n c t i o n o n T4 c o n c e n t r a t i o n in serum. O u r data agree with the findings of Renbour,n a n d of Walfish et al. i n the general description of the fluctuations b u t indicate that the variation in total serum T4 c o n c e n t r a t i o n is related to changes in the patient's posture. T h e data indicate the i m p o r t a n c e of p l a s m a v o l u m e alteration in d e t e r m i n a t i o n of d i u r n a l rhythms, particularly for substances circulating b o u n d to proteins. O u r observations emphasize the influence of p l a s m a dilution or c o n c e n t r a t i o n o n e v a l u a t i o n of the rate of disappearance of T4 f r o m p l a s m a a n d suggest a m e t h o d for correcting the scatter of individual values w h e n b l o o d sampling is n o t p e r f o r m e d u n d e r exactly similar conditions. MATERIALS AND METHODS One euthyroid normal subject ( J . J . ) , o n e patient with euthyroid Graves' disease (L.M.), two patients with moderate hyperthyroidism due to Graves' disease (A.D. and A.W.), and two moderately hypothyroid patients (J.N. and C.B.) were studied. They received a simultaneous intravenous injection of 40 #Ci of T4-1zlI and 10/zCi of 125I-labeled human serum albumin (12gI-RISA or Albumin-12gI). Thyroidal iodide recirculation was blocked in one patient (JJ.) by administration of potassium iodide, 100 mg daily. The protein bound lzlI (PBlaaI) and pB125I were measured 10 min, 30 rain, 60 min, 120 rain, 8 hr, and 24 hr after the injection, and then twice daily. After a uniform disappearance slope was attained for both PB13~I and PBlZSI, diurnal variations were studied by repeated sampling of blood at 8 a.m., 10 a.m., 1 p.m., 5 p.m., 9 p.m., and 2 a.m. During the period of frequent sampling, the PBlZ7I, hematocrit, and total plasma proteins (TP) were measured (using a T.C. Refractometer, American Optical Corporation, Buffalo, N. Y.) in addition to isotope labeled trichloroacetic acid (TCA) precipitable iodine. Under conditions of this study, determinations of PB131I values on replicate samples gave a standard deviation of less than 5% of the mean. pBleTI values were determined by Boston Medical Laboratory. The usual standard deviation on replicate assays of one sample in their laboratory is also under 5% of the mean. The maximal T 4 binding capacity of thyroxine binding globulin (TBG) and thyroxine binding prealbumin (TBPA),3 concentration of plasma free fatty acids (FFA),4 tyrosine,5 total thyroxine (TT4),6 and the T 4 dialyzable fraction (T4-DF)7 were also evaluated in some patients. Changes in 131I and 1251 activity in thyroid and liver were assessed by external counting over these organs using a Nuclear Chicago Scintillation Gamma Ray Counter. In the initial period patients were kept on a normal sleepwaking cycle (sleeping between 10:00 p.m. and 8:00 a.m.) and on a regular diet with meals at 8:00 a.m., noon, and 6:00 p.m. After completion of studies for a normal schedule, the patients were studied in various experimental conditions. Each subject served as his own control. In two subjects (J.N. and J.J.), meals were given as equal aliquots of food every 6 hr, while the patients were recumbent from 10:00 p.m. to 8:00 a.m. as usual; the 2:00 a.m. meal was consumed while the subject remained recumbent. In three subjects (J.L, C.B., and J.N.) the sleep-waking cycle was reversed. When awake patients were required to be up and about. Meals were scheduled at the usual time, but lunch was omitted. In three subjects (J.N., C.B,, and L.N.) the 17-hydroxy-corticosteroid and 17-ketosteroid excretion in the urines was studied during a normal sleep-waking cycle. Dexamethasone, .5 mg every 6 hr, was then given for 3 days while the patients were maintained on the usual sleep-waking cycle and the studies were repeated. The T4-125I plasma disappearance rate was evaluated in two obese patients (G.S. and
784
DE COSTRE ET AL. I
I
T
r
I
80
B
A.W (I. 1968
70 TOTAL
PROTEIN
(qm/1)
6C 5s 4O
HEMATOCRIT
(%)
30
THYROID- mI (% dose)
20
LIVER-13fl (% dose)
PB 1271 (p g ~
Fig. 1.--Typical study showing diurnal fluctuation of T4AslI and Albumin-12~I, hematocrit, total protein, and PB12q. N o diurnal fluctuation is apparent in thyroid and liver isotope content.
ALBUMIN -mini (% dose / 1)
PB '3'I (% dosell) 3
_ _
I
6:00 A.M.
]
l
12100 6 0 0 { RM
t 12:00 :
TI ME"
J
r
6:00 12:00 AM. } OF DAY
S,M.) during total fasting after injection of T4-131I and Albumin-125I. PBlalI, PB125I, sermn total proteins, thyroxine and dialyzable thyroxine fraction were measured in each sample, and the free T 4 concentration calculated. The slopes of the curves and the intercepts were evaluated by means of the method of least squares. RESULTS
In the normal pattern, diurnal variations occurred in all patients and were similar in direction. For this reason subsequent observations are presented without reference to thyroid diagnosis. Data for one period of frequent sampling (patient A.W.) is detailed in Fig. 1. The curves are given for a period of more than 24 hr. No consistent change is observed in thyroid and liver radioactivity, while the other parameters, except for tyrosine, show a similar modification. The maximal concentration of the various constituents in plasma occurred between 8:00 a.m. and 1:00 p.m., and the minimum level is observed around 2:00 a.m. The tyrosJne concentration obtained in two patients (A.W. and A.O.) and the free fatty acids in one patient (A.O.) did not follow the same rhythm. The PBI~II curves obtained for each patient during a normal sleep cycle are given in Fig. 2. The values are expressed as a percentage of the 2:00 a.m. values. The seven curves show the same general shape and their mean value is
DIURNAL RHYTHM
Fig. 2.--Diurnal fluctuations of plasma T4-131I for seven different periods in six patients. Concentrations are expressed as per cent of 2:00 a.m. value on second day taken as 100%. Thin lines: individual patient's data. Thick line: mean for all observations. The 11% difference between 2:00 a.m. values on two consecutive days is due to T4 degradation. Mean fractional degradation for all patients is 10.6%/day.
785
130
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,
DAY
characterized by a maximal PB~II level between 10:00 a.m. and 1:00 p.m., and a minimal concentration at 2:00 a.m. There is on the average a 19% difference between the maximum value at 10:00 a.m. and the minimum at 2:00 a.m. The mean plasma T4-1a31 disappearance slope is .106 fraction per day for the six patients. On average the biologic decay of thyroxine 1~1I would account for 16 h r / 2 4 hr )< 0.11 fr./day X 19% = 8% of this decline. Thus the decrease in PBIZlI levels from 12:00 noon to 2:00 a.m., attributed to the diurnal rhythm, is on average 11% of the noon value. A study of the reversal of the sleep-waking cycle was performed in four patients; a typical result is shown in Fig. 3. It is evident that the usual pattern is demonstrated for the normal sleep-waking cycle. As soon as the patient's sleep cycle is reversed, there is a reversal in the rhythm of the TP, T4-1alI and AlbuminJ25I concentrations in plasma. The minimal concentratio.n, at 2:00 a.m. for the normal cycle, is altered to a minimal concentration at 2:00 p.m. during the reversed sleep schedule. The minimal concentration corresponds i.n each case to the recumbent position. The influence of changing the feeding cycle was studied in three patients given four equal feedings at 6-hr intervals. The diurnal rhythm minimum was at 2:00 a.m. there was ,no change in the shape of the curve. The influence of the adrenal steroid rhythm was analyzed in three patients; a typical study is presented in Fig. 4. The urinary 17-hydroxycorticoid excretion demonstrated the usual diurnal variation during the sleeping and the waking period. After suppression of adrenal function by 2 m g / d a y of dexamethasone, there was no change in the rhythm for total protein, PB"~[, or AlbuminYSI in plasma. The practical importance of postural changes in the evaluation of T4 and
DE COSTRE ET AL.
786 Ioo
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Fig. 3.mInfluence of posture on plasma concentrations of T4-131I, Albumin-125I, and total proteins. Thick line: periods of recumbency. Reverse sleep cycle values (circles) for Albumin-125I and T~-131I are at higher level than normal sleep cycle values (triangles) because of gradual biologic decay of labeled compounds. Daytime sleep pattern reverses usual diurnal cycle and causes low values at noon. albumin metabolism is demonstrated in Fig. 5. This obese subject was on a total fast for 1 wk prior to and during the study. During the 12 days of this study, blood was taken each morning before the patient got up, except on days 6 and 10. On these days there was a distinct increase in plasma protein concentration. The slopes of the T4-13lI and Albumin-125I disappearance curves, plotted by the method of least squares, for this data are given in lines A and A', Fig. 5. When the albumin-125I and T4-a3H values are related to the protein concentration, deviations from the straight line become ne~igible, and the slopes have slightly different values (lines B and B', Fig. 5). Differences of 7% and 33%, respectively, were found for T4 and albumin slopes, aaad differences of 4% and 17%, respectively, for T4 and albumin distribution spaces. TBG and TBPA capacity, T4, and dialyzable fraction were determined on samples obtained from the same patient. It may be seen that absolute flee T4 remained relatively constant despite significant variation of total T4 concentration (Table 1), and
DIURNAL RHYTHM
787
that variations in total T4, TBG, and TBPA were directly proportional, and the T4 dialyzable fraction inversely proportional to the total serum protein concentration. Similar changes were observed in a thyrotoxic patient (A.O.) during one complete diurnal cycle (Table 2). DISCUSSION When frequent blood samples are analyzed during a study of the disappearance of labeled T4 from plasma, a daily maximum plasma concentration of radioactive PBI is observed between 10:00 a.m. and 2:00 p.m., while a miniI
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Fig. 4.--Influence of adrenocortical secretion on plasma concentration. Left panel represents normal pattern with normal diurnal variations of 17-hydroxycorticoid excretion. Right panel shows maintenance of rhythm after suppression of adrenocortical secretion with dexamethasome. Four days elapsed between first and second studies. Dexamethasone does not influence rhythm.
788
DE COSTRE ET AL.
8 ~
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DAYS
Fig. 5 . - - I m p o r t a n c e of postural changes in evaluation of T4 and R1SA metabolism. Line A: data on T4-1311% dose/1; ten, related in line B to protein content of samples (% dose • 10 g T P ) . Line A ' : data on Albumin-levi % d o s e / l , related in line B' to protein content of samples (% dose • 10 g T P ) . Boxes identify corresponding half-times ( t 8 9 decay rates ( K ) , and distribution spaces (D.S.). m u m occurs a r o u n d 2 : 0 0 a.m. T h e la b e le d h o r m o n e c o n c e n t r a t i o n at 2 : 0 0 a.m. is on average 1 1 % b e l o w the 1 0 : 0 0 a.m. value. N o consistent reciprocal cha,nges were o b s e r v e d in liver T4 content, i n d i c a t i n g there was n o m o v e m e n t of h o r m o n e
Table L--Influence of Posture on Serum Total Proteins, T 4 Binding Proteins, and T4 Concentration D a y of Sampling
Position
4 recumbent 5 recumbent 6 standing 9 recumbent 10 standing % change from days 4 t o 6 % change from days9 to 10
Total Protein (g/100 ml)
Total T, Dialyzable Free Thyroxine Fraction Thyroxine (/Zg/100 ml) (%) (m/tg/100 ml)
0.056 0.0.57 0.047 0.052 0.040
3.36 3.28 3.29 3.07 3.16
T4 Binding Capacity TBG* TBPA (#g/100 ml)
6.2 6.2 7.3 6.3 7.8
6.0 5.7 7.0 5.9 7.9
21.4 20.2 24.5 21.5 25.4
• • • • •
0.9at 0.2 a 0.3 b 1.0a 0.9 b
116.2 102.8 132.0 120.2 146.7
+17.7
+16.7
-16.1
-2.1
+14.7
+13.6
+23.8
+33.9
-23.1
+2.9
+18.4
+22.1
* Mean _ standard deviation on four consecutive determinations of same sample. t Values not significantly different from each other are indicaed with same superscript. Those with different superscripts are significantly different at p<0.05 to p<0.001.
DIURNAL RHYTHM
789
Table 2 . - - D i u r n a l Variation of Thyroxine Concentration and Plasma Constituents in Patient A.O. With Thyrotuxicosis D u e to Graves' Disease Time of Day 7 : 30 10:00 5:00 2:00
a.m. a.m. p.m. a.m.
Total Protein (g/100 ml)
PB127I (#gI/100 ml)
6.4 7.1 7.2 6.3
13.2 14.4 15.2 13.0
T~ Dialyzable Free TI Binding Fraction Thyroxine Capacity of TBG Hematocrit (%) (m/xgTa/100 ml) (~tg/100 ml) (%) 0.097 -0.091 0.113
12.8 -13.8 14.7
16.3 20.0 21.1 16.2
43.0 48.5 44.5 40.0
in and out of this storage area. However, a change in this parameter might be too small to be detected by the external counting technique employed in our study. The cycle described for T4-131I (PBla~I) was paralled exactly by changes in Albumin-12~I, PB~27I, total thyroxine, total protein concentration, TBG and TBPA capacity, and hematocrit. Reversed cyclic changes were observed in the T4 dialyzable fraction, resulting in a stable free T4 concentration. The cyclic changes in serum T4 concentration were not influenced by the feeding rhythm nor by suppression of adrenal secretion. When the sleep-waking cycle was reversed, there was an immediate reversal of the rhythm with a minimum corresponding to the new sleeping period. This rhythm is similar in both degree of change and in periodicity to that described by Doe for plasma cortisone-binding globulinY Since Albumin-12~I leaves the intravascular space relatively slowly and is not accumulated in any organ, marked and cyclical changes in its concentration in blood are presumably good indications of plasma volume shifts. The existence of a rhythm for AlbuminJ2aI concentration, similar to that for T4, suggests that the changes observed are mostly due to diurnal variations in plasma volume, with reciprocal changes in plasma protein concentration. A redistribution of fluids in the body occurring with a change from the recumbent to the upright posture has long been recognized,TM and could explain the rhythm noted in these studies. Hydrostatic pressure in the lower part of the body is higher during upright standing than during recumbeny, and the equilibrium with the intravascular osmotic pressure is altered. According to this mechanism, upon upright standing there is a transfer of fluid to the extravascular space, which increases the concentration of nondiffusible plasma constituents until a new equilibrium is reached between two pressures. This phenomenon could explain the rhythm observed for hematocrit, PB~27I, and TT4 concentration, TBG capacity, and the relative constancy of the free T4 concentration. It is not necessary to hypothesize that T4 or TBG moved in and out of the vascular compartment, or that variations in T4 secretion or metabolism occurred. Cyclic hemodilution and hemoconcentration, by transfer of water, could explain the rhythm we have observed. The well-described diurnal cycle in urine flow is characterized by peak flow from 6:00 to 12:00 a.m., a decrease in the afternoon, and nadir between 12 midnight and 6:00 a.m21 This cycle is also reversed by a "reversed" sleep cycle. A similar rhythm is seen in patients kept recumbent throughout the 24-hr period32 This cycle could possibly play a causative role. in the cyclic variations of plasma protein concentration we have noted, although data are not sufficient to establish this relationship.
790
DE COSTRE ET AL~
It has been suggested1~ that muscular exercise increases osmotic pressure in muscle. Muscular work associated with upright posture could represent a second factor influencing transfer of fluids from intra- to extravascular compartments. The three mechanisms we have noted are believed to be sufficient to explain the rapid change in TT4 concentration when subjects get up in the morning, but do not adequately explain the tendency for TT4 concentration to resume a lower level in the afternoon. It is possible that other undetected regulatory mechanisms may be involved and may tend to buffer swings in concentration toward a "normal" value. Neither changes in the feeding rhythm nor suppression of adrenocortical secretion by dexamethasone were able to affect the T4 rhythm. This latter observation suggests that, at least for the short periods studied, the fluid transfers that we believe mediate the rhythm are not dependent upon cyclic variation in adrenal glucorticoid secretion. A diurnal rhythm has been reported for plasma amino acidsp ,~4 and concentration of tyrosine, phenylalanine ,and tryptophan being lower around 2:00 a.m. and higher around 10:00 a.m. For some amino acids the changes were 1.2 times the basal value, a level comparable to concentratio.n changes detected in our study. However, these amino acids are not bound tightly to protein, so this "rhythm" is presumably not caused by the same mechanism we postulate for the thyroxine rhythm. Evaluation of any metabolic rhythm must take into account the cyclic changes in the concentration of nondiffusible plasma constitutents, which could potentially interfere with the phenomenon studied. It is interesting to note that 11% change in the PBI can be related simply to the chronology of plasma sampling. The same conclusion is valid for other nondiffusible plasma constituents such as proteins or red blood cells. Modifications of that range may be completely independent of metabolic alterations. The induction of anesthesia by ether also results in a transient increase in plasma PBI concentration15 which may be related to changes in plasma volume. ~6 A last corollary of these observations is the importance of accurate standardization of the conditions of sampling for PBa27I and PBa3H determi~nations, and as a matter of fact, for any protein-bound compound in plasma. In the case of T~ disappearance rate evaluation, the scatter of the individual values, when other technical factors have been excluded, may be reduced by sampling under reproducible enviro,nmental conditions, or by correction of the radioactive PBI values for plasma protein concentration in the test sample. A reduction in the spread of the observations may lead to a greater accuracy of turnover data. The present investigation confirms the existence of a diurnal rhythm in the concentration of serum total thyroxine and certain other plasma constituents. The rhythm is related to a large extent to passive movement of fluids into and out of the vascular compartment. These diurnal variations are not related to food intake nor to adrenocortical function, but are influenced by the patient's posture. Diurnal variations in thyroid hormone secretion, a7 metabolism, or accumulation in liver may occur, but are not required to account for the fluctuations we have observed. The variations we observed in the total thyroxine concentration in serum are associated with constant concentration of free thyroxine, as would be expected from the known constancy of free thyroxine during dilution of
DIURNAL RHYTHM
791
plasma, is Since free thyroxine is considered to be the active h o r m o n a l form i n serum, capable of traversing cellular m e m b r a n e s a n d activating m e t a b o l i c processes, the d i u r n a l r h y t h m observed for s e r u m total thyroxine m a y n o t b e associated with a similar r h y t h m in intracellular h o r m o n a l c o n t e n t a n d degradation. Nevertheless, these r h y t h m s m a y b e a very i m p o r t a n t cause of scatter in the observations recorded during T4 t u r n o v e r studies if b l o o d s a m p l i n g is n o t perf o r m e d u n d e r the same conditions. A correction m e t h o d is p r o p o s e d to reduce the spread of observations a n d to increase the accuracy of the metabolic studies. ACKNOWLEDGMENT We are grateful to Mr. Richard J. Wurtman, Department of Nutrition and Food Sciences, Massachusetts Institute of Technology, Boston, Mass., for the determination of tyrosine, and to Dr. Philip Felig and Dr. George F. Cahill, Joslin Research Laboratory, Boston, Mass., for the determination of free fatty acid and permission to study the two obese patients during fast. REFERENCES 1. Renbourn, E. T.: Variation, diurnal and over longer periods of time, in blood haemoglobin, haematocrit, plasma protein, erythrocyte sedimentation rate, and blood chloride. J. Hygiene 45: 455, 1947. 2. Walfish, P. G., Britton, A., Melville, P. H., and Ezrin, C.: A diurnal pattern in the rate of disappearance of la~I-labeled 1-Thyroxine from the serum. J. Clin. Endocr. 21: 582, 1961. 3. Refetoff, S. and Selenkow, H. A.: Familial thyroxine-binding globulin deficiency in a patient with Turner's syndrome (XO) : genetic study of a kindred. New Eng. J. Med. 278: 1081, 1968. 4. Trout, D. L., Estes, E. H., Jr., and Friedberg, S. I.: Titration of free fatty acids of plasma: study of current methods and a new modification. J. Lipid Res. 1: 199, 1959. 5. Wurtman, R. J., Rose, C. M., Chou, C., and Larin, F. F.: Daily rhythms in the concentrations of various amino acids in human plasma. New Eng. J. Med. 279: 171, 1968. 6. Murphy, B. E. P., and Jachan, C.: The determination of thyroxine by competitive protein binding analysis employing an anion exchange resin and radiothyroxine. I. Lab. Clin. Med. 66: 161, 1965. 7. Fang, V. S., and Selenkow, H. A.: Determination of free thyroxine in serum by low-temperature equilibrium dialysis. Clin. Chem. 16: 185, 1970. 8. Reddy, W. J.: Modification of the Reddy-Jenkins-Thorn method for the esti-
mation of 17-hydroxycorticoids in urine. Metabolism 3: 489, 1954. 9. Doe, R. P., Fernandez, R., and Seal, U. S.: Measurement of corticosteroid-binding globulin in man. J. Clin. Endocr. 24: 1029, 1964. 10. Fawcett, I. K. and Wynn, V.: Effects of posture on plasma volume and some blood constituents. J. Clin. Path. 13: 304, 1960. 11. Smith, H.: The Kidney New York, Oxford University Press, 1951, p. 339. 12. Sirota, J. H., Baldwin, D. S., and Villarreal, H.: Diurnal variations of renal function in man. I. Clin. Invest. 29: 187, 1950. 13. Welt, L. J., Orloff, J., Kydd, D. M., and Oltman, J. E.: An example of cellular hyperosmolarity. I. Clin. Invest. 29: 935, t950. 14. Wurtman, R. J., Chou, C., and Rose, C. M.: Daily rhythm in tyrosine concentration in human plasma: Persistence on low protein diets. Science 158: 660, 1967. 15. Fore, W., Kohler, P., and Wynn, I.: Rapid redistribution of serum thyroxine during ether anesthesia. J. Clin. Endocr. 26: 821, 1966. 16. Albert, S. N.: Blood Volume. Springfield, II1., Thomas, 1963, p. 54. 17. Nicoloff, J. T.: A new method for the measurement of thyroid iodine release in man. J. Clin. Invest. 49: 1912, 1970. 18. Oppenheimer, J. H. and Surks, M. I.: Determination of free thyroxine in human serum: a theoretical and experimental analysis. J. Clin. Endocr., 24: 785, 1964.