Thyroid Metabolism of Chickens

Thyroid Metabolism of Chickens 1. E S T I M A T I O N O F H O R M O N E C O N C E N T R A T I O N BY T H E T H Y R O X I N E B I N D I N G GLOBULIN TECHNIQUE J. D . M A Y 1 , L. F . K U B E N A 1 , J. W. D E A T O N 1 AND F. N .

REECE2

United States Department of Agriculture, A.R.S., State College, Mississippi

39762

(Received for publication July 20, 1972)

POUXTRY SCIENCE 52: 688-692, 1973

I

N mammals, protein bound iodine is a common criterion of measurement of thyroid hormone metabolism. For chickens, however, Mellen and H a r d y (1957) found t h a t protein bound iodine values where not significantly changed b y either cold stress or thiouracil treatment. This demonstrated t h a t the protein bound iodine technique was not a satisfactory measurement of thyroid hormone metabolism for chickens. T h e failure of protein bound iodine to accurately estimate thyroid function m a y be due to the absence of thyroxine binding globulin in chicken blood as shown b y T a t a and Shellabarger (1959). Little information is available concerning the normal levels of thyroxine in chicken plasma. Controversy exists for values of thyroxine t h a t are reported for chickens. Refetoff et al. (1970) measured thyroxine concentrations using a thyroxine binding globulin technique and re1

Animal Science Research Division, Poultry Research Branch, South Central Poultry Research Laboratory, State College, Miss. 2 Agricultural Engineering Research Division, Farm Electrification Research Branch, South Central Poultry Research Laboratory, State College, Miss.

ported values of 1.4 and 1.6 jug. per 100 ml. serum for adult chickens. Sadovsky and Bensadoun (1971) reported diurnal variation in the thyroxine values of t h e rooster ranging from 3.6 to 5.6 /ug. per 100 ml. plasma. Their technique involved use of a cation exchange resin and thin-layer chromatography with chemical determination of iodine. This paper describes a technique for estimating the plasma thyroxine concentration for chickens and reports the concentration in plasma for chickens ranging from 13 days old to maturity. Thyroxine recovery and interference by triiodothyronine are reported. MATERIALS AND

METHODS

Procedure: T h e thyroxine binding globulin (TBG) procedure used in these studies was developed from the procedure described b y M u r p h y (1965). Plasma was treated with two volumes of ethanol and the extract was clarified b y centrifugation. Duplicate 0.3 ml. samples were evaporated to dryness by a stream of air in a water b a t h at 45° C. T B G solution (1 ml.) at 4° C. was added t o each test t u b e and the racks of test

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ABSTRACT The thyroxine binding globulin technique was used to estimate T4 concentration in the plasma of chickens from 13 days of age to maturity. Average T 4 values ranged from .69 to 1.21 jug. percent, not corrected for percent recovery. T4 recovery averaged 44 percent. T4 values were lowest for mature chickens. The optimum concentration of serum and T4 in the thyroxine binding globulin solution was 1.8 percent serum and 74 ng. percent T4. Excess serum or T4 caused a reduction in the sensitivity of the assay. These results are in reasonable agreement with a previous report of T4 concentration using the thyroxine binding globulin technique, but are substantially lower than the reported results obtained by cation exchange resin and thin-layer chromatography.

THYROID METABOLISM

Thyroxine assays were conducted on plasma from commercial broiler type chicks 13, 15, 40, and 60 days of age and from mature breeding stock 3 . T h e 13- and 40-day-old chicks were in environmental chambers at a constant 32.2° C , and the 15-day-old chicks received supplemental heat from heat lamps. T h e 60-day-old broilers and the adult chickens were maintained without supplemental heat. Standard Solutions: A weighed q u a n t i t y (5-10 mg.) of L-thyroxine sodium pentah y d r a t e was dissolved in 1 ml. of 0.1 N N a O H and the resulting solution was diluted to a concentration of 25 fig. thyroxine (T4) per ml. with 95 percent ethanol. A working T4 standard solution was prepared b y diluting the above solution 1:5000 with 95 percent ethanol. T h e standard curve was prepared b y pipetting 0.1, 0.2, 0.3, 0.4 and 0.5 ml. volumes of the working solution into test tubes and evaporating to dryness. Standard solutions of 3,5 diiodo L thyronine (T 2 ) and 3,5,3' triiodo L thyronine (T3) were also prepared in 95 percent ethanol. Standard solutions were assayed b y the eerie a m m o n i u m sulfate technique to verify accuracy of preparation. 3

Samples from mature breeding stock were obtained through the courtesy of Prof. Lester J. Dreesen, Poultry Science Dept., Mississippi State University.

Barbital Buffer: ( p H 8.6, ionic strength .075) Barbital sodium (15.464 g.) was dissolved in 800 ml. water. Barbital (2.823 g.) was added, dissolved, and diluted to bring the volume to 1000 ml. TBG Solution: H u m a n serum 4 6 ml., 5-20 microcuries T 4 -I-131 6 (containing approximately 0.4 ng. T 4 ), 5 ml. 1 percent phenol, and 5 ml. propylene glycol, were thoroughly mixed and diluted with barbital buffer to 500 ml. Resin: Rexyn 202 6 resin was soaked in barbital buffer overnight. Excess buffer was discarded and the resin was dried at 105° C. to drive off moisture. Ti Recovery: T 4 recovery was determined by pipetting 0.5, 1.0, 1.5, and 2.0 ng. quantities of T 4 into test tubes and evaporating the 95 percent ethanol. E a c h tube then received 0.5 ml. of pooled plasma and was incubated at 37° C. for 30 minutes. T h e T4 assay was then conducted as for other samples. T 4 values reported were not corrected for percent recovery. RESULTS AND DISCUSSION The estimation of T 4 concentration in chicken plasma using the T B G technique with h u m a n serum is based on the binding of T4 b y h u m a n serum. Displacement of labelled T 4 from the T B G solution b y T 4 analogues biases the assay upward to the extent t h a t the analogue is present and is bound b y the serum used in the T B G solution, Results obtained (Table 1 and Figure 1) show t h a t h u m a n serum binds T 3 and T4 b u t not T 2 . T 4 was more efficient t h a n T 3 a t displacing T 4 -I-131 frorn the T B G . T h e T 4 equivalent in Table 1 was obtained b y determining from t h e T4 curve the q u a n t i t y of T4 required to get a reduction in counts equivalent to the re* Code 5578-57-3 Human serum, desiccated; Difco Laboratories. 6 6706 Thyroxine 1-131; Abbott Laboratories. • Fisher Scientific Company.

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tubes were placed in a water b a t h at 45° C. for 8 minutes. T h e racks were shaken to dissolve any residue in the test tubes and moved to a water bath at 4° C. After at least 10 minutes pre-weighed 500 mg.quantities of resin were quickly added to each tube. Each rack of test tubes was then shaken vigorously for 1 minute and returned to the cold water bath. Barbital buffer (3 ml.) was added to each tube and after thorough mixing, 1.0-ml. samples were removed and counted in a liquid scintillation spectrophotometer.

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J. D. MAY, L. F. KUBENA, J. W. DEATON AND F. N. REECE

TABLE 1.—Relative binding of Tt, T3 and Tt by human serum Quantity (nanograms) 0

35,972*

T4 T3 Tt equivalent T2

28,315 32,915 .35** 36,201

23,689 30,357

21,005 28,796

.70

18,776 26,522 1.38 35,281

.93

36,080

35,681

17,001 25,115 1.67 34,862

* Disintegrations per minute. ** Determined from the T< curve.

Effectiveness =

T4 equivalent T 3 concentration

xioo

Effectiveness for the 1, 2,3,4, and 5 levels of T 3 were 35, 35, 31, 34, and 33, respectively. This indicates that T 3 is about onethird as effective as T4 at displacing T41-131 from the TBG. These results differ from the report of Dalrymple and Utiger (1970) only in that the relative potency of

T3 was one-fifth that of T4 in their test rather than the one-third that we obtained. If T 3 and T4 were present in equal quantities in chicken plasma, the T4 determination could be expected to be as much as onethird greater than actual. Wentworth and Mellen (1961) suggested a 0.67 ratio of T 3 to T4 and did not find T 2 in chicken plasma. Sadovsky and Bensadoun (1971) assayed rooster plasma collected at 4-hour intervals over a 24-hour period and found

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1. Standard curves for T 2 C |) and T, ( • T,

FIG. 2. T4 standard curves as affected by TBG solution composition ( B ™ - H = 1-2% serum, 536 ng. percent Ti; Jt A = 1-2% serum, 74 ng. percent T<; • • — • # = 1.8 percent serum, 74 ng. percent T«).

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duction caused by T 3 . The relative effectiveness of T 3 versus T 4 may be described by:

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THYROID METABOLISM

TABLE 2.—Effect of serum and thyroxine 1-131 concentration on thyroxine quantitation Thyroxine: binding globulin solution Thyroxine standard, ng. 0.5 1.0 2.0 3.0 4.0 5.0

1.2% serum 536 ng.% thyroxine 101.8* 99.5 97.9 98.4 94.0 92.2

1.2% serum 1.8% serum 74 ng.% 74 ng.% thyroxine . thyroxine 94.0 93.4 95.2 87.6 85.0 76.3 78.5 69.4 77.8 61.9 75.9 56.5

TABLE 3.-—Recovery of Tifrom plasma T 4 Added (ng.)

T< Assay (ng.)

Recovery

Pooled Plasma 0.5 1.0 1.5 2.0

0.60 0.89 1.02 1.22 1.32

58 42 41 36

%. .

TABLE 4.—Plasma thyroxine concentration* of chickens Sex Male 13 15 40 60

days days days days

Adult Trial 1 Trial 2

Mixed

Female .92±.12(9) 1.21 ± . 1 4 (10)

1.15 ± . 1 2 (10)** 1.21 ± . 2 0 (10) 1.38±.23(10) 1.05±.05(14) .71 ± . 0 7 (8) .84±.12(5)

1.00±.06(14) .69±.07(8) .93 ± . 2 7 (10)

* tig. per 100 ml. ** Mean ± standard error (No. chickens).

an average ratio of 1.67 for T3/T4. T h e effect of varying the level of h u m a n serum or T4 in the T B G solution is illustrated in Figure 2 and the data are given in Table 2. T h e optimum concentrations of serum and T4 were 1.8 percent and 74 ng. percent, respectively. T h e proper ratio of T4 to serum is important in obtaining satisfactory standard curves. T h e result is understandable since T 4 -I-131 in the T B G solution competes with sample thyroxine for binding sites in t h e case of excess T4. I n the case of excess serum, sample T 4 would be bound b y serum without displacing T4-I-131. Results of determination of recovery of T4 from plasma are presented in Table 3. Recovery averaged 44 percent which is considerably lower than t h a t reported b y -Dalrymple and Utiger (1970). T4 levels in chickens of various ages

were determined and are-presented in Table 4. Plasma T 4 levels were not consistently affected by sex, b u t m a t u r e chickens had lower thyroxine levels t h a n growing chickens. T h e range of thyroxine values obtained were in the same range of values reported b y Refetoff et al. (1970). REFERENCES Dalrymple, D. E., and R. D. Utiger, 1970. A micromethod for the determination of serum thyroxine: Studies of serum throxine concentrations and thyroxine binding in rat serum. J. Lab. Clin. Med. 75:325-335. Mellen, W. J., and L. B. Hardy, 1957. Blood protein-bound iodine in the fowl. Endocrinol. 60: 547-551. Murphy, B. P., 1965. The determination of thyroxine by competitive protein-binding analysis employing an anion-exchange resin and radiothyroxine. J. Lab. CHn. Med. 66:161-167. Refetoff, S., N..J. Robin and V. S. Fang, 1970. Parameters of thyroid function in serum of 16 selected vertebrate species: A study of PBI,

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' Percent of the counts without added thyroxine standard.

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J. D. MAY, L. F. KUBENA, J. W. DEATON AND F. N. REECE

serum T4, free T4, and the pattern of T 4 and T 3 binding- to serum proteins; Endocrinol.1 86: 793805. Sadovsky, R j; and A. Bensadoun, 1971. Thyroid iodohormones in the plasma of the rooster (Gallus domesticus). Gen. Comp. Endocrinol. 17: 268274.

Tata, J. R., and C. J. Shellabarger, 1959. An explanation of the difference between the responses of mammals and birds to thyroxine and triiodothyronine. Biochem. J. 72: 608-613. Wentworth, B. C ; and W. J. Mellen, 1961. Circulating thyroid hormones in domestic birds. Poultry Sci. 40: 1273-1274.

.WILLIAM C. REINKE 2 , JOHN V. SPENCER AND LYDIA J. TRYHNEW 3 Department of Animal Sciences, Washington State University, Pullman, Washington 99163 (Received for publication July 21, 1972)

ABSTRACT Various differences between hard cooked, difficult-to-peel eggs and easy-topeel eggs were studied. Experiments included artificial thinning of the albumen, measurement of albumen viscosity, accelerated increase or decrease of albumen pH, exchange of fresh and old egg contents and microscopic observation of the shell membrane. Old eggs exposed to a carbon dioxide atmosphere exhibited a decrease in albumen pH, an increase in the viscosity of adhering albumen and were difficult to peel. Fresh eggs exposed to ammonia exhibited an increase in albumen pH, no increase in the viscosity of adhering albumen and were easy to peel. Albumen was thinned by soaking in various chemicals without changing the albumen p H significantly. Low p H values, i.e. below 8.9 to 8.7, corresponded to low peeling quality regardless of the amount of albumen thinning. Further evidence for the importance of albumen p H on peeling quality was obtained when the contents of fresh and old eggs were exchanged and then hard cooked. Microscopic observations revealed that old egg shell membrane was compact and that a wide-ovomucin border was present between the inner membrane and adhering albumen. Shell membranes of fresh eggs were less compact and a narrow, less concentrated ovomucin border was observed. Results obtained suggested that albumen pH affected the shell membranes and adhering albumen directly to influence peeling quality. POULTRY SCIENCE 52: 692-702, 1973

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A R D - C O O K E D , f r e s h l y l a i d eggs a r e difficult t o p e e l c l e a n l y w i t h o u t t e a r i n g t h e a l b u m e n . A s a n egg ages d u r ing storage, certain physical and chemical c h a n g e s o c c u r i n t h e c o m p o s i t i o n of t h e egg a n d t h e e a s e of p e e l i n g i n c r e a s e s . I t h a s b e e n o b s e r v e d t h a t t h e p H of t h e a l b u m e n rises f r o m 7.6 t o as h i g h as 9.7 a c c o m p a n i e d b y a b r e a k d o w n in t h e thick 1

Scientific Paper No. 3566, College of Agriculture, Washington State University, Pullman. Project No. 1325. 2 Present address: ; Checkerboard Foods Division, Ralston Purina, St. Louis^ Missouri. • Present address: Iiydia Tryhnew Goatcher, Department of Dairy Science, University of Maryland, College Park; Md.2074(J. •••"•'•

albumen structure (Romanoff and Romanoff, 1949). Swanson (1959a) indicated that the change in pH of albumen is related to ease of peeling. Above pH 8.6 to 8.7, he found that little or no difficulty in peeling was experienced. Similarly, Fuller and Angus (1969), in their study of the pH of both uncooked albumen and homogenized albumen from hard cooked eggs indicated that the "crossover" from poor to good peeling characteristics corresponded to pH values of 8.6 to 8.9 of raw egg white. Swanson (1959b) also reported that freshly laid eggs could be made to peel easily if they were exposed to ammonia fumes until the pH of the albumen

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The Effect of Storage Upon the Chemical, Physical and Functional Properties of Chicken Eggs 1