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1952 Biochemical Studies on Anolis Carolinensis. Herbert Clay Dessauer Louisiana State University and Agricultural & Mechanical College
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Recommended Citation Dessauer, Herbert Clay, "Biochemical Studies on Anolis Carolinensis." (1952). LSU Historical Dissertations and Theses. 8019. https://digitalcommons.lsu.edu/gradschool_disstheses/8019
This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. BIOCHEMICAL STUDIES ON ANOLIS CAROLXNENSIS
A Dissertation
Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy In The Department of Biochemistry School of Medicine
*7 Herbert Clay Dessauer B. S., Louisiana State University, 1949 June, 195^ UMi Number: DP69397
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LOUISIANA. STATE UNIVERSITY LIBRARY - ^
ACK NOWLEDGMEMT
I am Indebted to many persons for aid and cooperation In the courae of the research and preparation of this paper* Mrs* Marlon Otvell of the library ataff gave Invaluable assistance In locating many scarce references* The staff of the Art Department of the Medical School produced the photographs* My wife and Mrs* Evangeline Gaglian© helped in the typing of the manuscript* Miss Lucy Qremlllion contributed patient assistance In analyses In which an extra pair of hands was required* To Dr* Roland A* Ceulson, my major professor, X am grateful for advice, guidance and, especially, the benefit of his enthusiasm* Finally, X thank Dr* Brasda and the entire staff of the Biochemistry Department for instruction and friendly criticism* 11
37 £74 L 9 3 o i
I 3 5 Z
C -Z- 355422 TABLE OP CONTENTS
I. INTRODUCTION 1 II. SURVEY ANALYSES 1 . Blood Collection and handling 4 2. General blood studies 5 3 • Determination of blood and plasma electrolytes 7 4. Blood and plasma nitrogen studies xo 5. Blood sugar 10 6. Urinary constituents 12 7. Fecal analysis 14 S. Composition of the animal ash 13 9. Discussion and analysis of data 16 III. METABOLIC STUDIES 1. Introduction 21 2. Analytical routine 22 3. Seasonal glucose and liver studies 26 A* Seasonal lipid studies 30 5 . Studies on lipid and glycogen distribution 34 6 . Seasonal respiration studies 36 7. Summary of seasonal metabolism 39 I?. BIBLIOGRAPHY 43 V. APPENDIX I 53 VI. APPENDIX II 54 VII. APPENDIX III 56 VIII. VITA 60 ill TABLES
1. Erythrocyte Studies 7 II. Blood Electrolytes 9 III. Blood Nitrogenous Constituents 10 IV. Comparison of tbs Results of Two Methods of Determining the Reducing Substance in the Blood 11 V* Urine Electrolytes 13 VI. Urine Nitrogenous Constituents n VII. FeeMl Analyses 15 VIII. Ash Analyses on Specimens of Anolls oarolinensle 17 IX. Seasonal Blood Clucoae 27 X. Croup Blood Clueose 2? XI. Seasonal Liver Studies 28 XII. Croup Liver Studies 28 XIII. Seasonal Lipid Studies 32 XIV. Croup Lipid Studies 32 XV. Pigment Studies 33 XVI. Distribution of Clycogen in the Liver and in the Hepateetoxaised Carcass 37 XVII. Distribution of Lipid in the Liver and in the Hepateetomised Carcass 37 XVIII. Qxy&en Consumption of Anolia oarolinensis 38 XIX. Respiratory Quotient of Anolls carollnensls 38 iv FIOTOES
1* Blood Centrifuging 6 2* Water Distribution IS 3* He spirometer 24 4« Liver Weight as per cent Body Weight 29 $. Liver Weight per unit Body Weight 31
6 « Liver Analysis Anolls earollnensis 35 7* Seasonal Variations Anolls earollnensis 40 v ABSTRACT
The biochemistry of reptiles is a large and practically unexplored field of investigation. Among reptiles the liaard, Anolls carolinensia. is plentiful, inexpensive and has many other advantages as an experimental animal. Although this lis&rd la very small, blood and urine analyses can be made readily with modern methods of micro analysis* Techniques vere devised for the collection and handling of blood. The nitrogenous composition of the blood and urine vaa determined. Fairly complete analyses of the inorganic substances of the blood, plasma, urine, feces and the total ash vere made, end the distribution of water in the animal was calculated from the experimental data. These studies indicate that lisards are more acidic than either alligators or turtles, and that Anolls has a higher plasma chloride (127 meq./L.) and phosphorus (2*6 mM./L.} and a lover average blood pH and carbon dioxide content (15 mM./L.) than mammals. Throughout 1951 analyses were made each month on Anolls to compare the effects of the seasons on animals obtained vl fresh from the wild with groups kept under laboratory condi tions of constant temperature and controlled hours of light* These analyses Included blood glucose, liver glycogen, total body lipid and water, oxygen utilisation and R. Q* Glucose, liver weight, liver glycogen and total lipid vary seasonally* There are no significant seasonal variations in the K* Q, and the oxygen requirement of the animal* In the late summer and fall the body weight and to a greater extent the liver weight increase due to the storage of glycogen and fat* With the increased storage of liver glycogen the blood glucose falls to a low level (100 mg. per cent). By November the lipid and carbohydrate content of the body represent close to 5 $ kilocalories per 100 grams of animal* Only three per cent of this reserve energy, however, is due to carbohydrate. In the winter and spring the blood sugar rises to a maximum of nearly 200 mg* per cent, and the liver glycogen and total lipid fall as the animal uses its body stores* By May these stores represent only one sixth of the available energy content of the animal in November* Neither varying the hours of light to which the animals were exposed nor keeping the animals at a constant temperature throughout the year had any marked effect upon the body constituents analysed* vii INTRODUCTION
Although those mammals of general Interest to medical biochemistry have been studied In detail for some fifty years, comparative biochemistry as a field of study is little more advanced than was the field of comparative anatomy a hundred years ago* It is curious to note that a century old knowledge of the anatomy of the most common reptiles of the world has not stimulated research in the chemical composition of this important class of animals* Appendix II, which summarizes an exhaustive search of the literature on studies upon reptilian blood, indicates the sparsity of information* The reptiles, thus, offer a wide and largely unexplored field of investigation in practically every phase of biochemistry* Among REPTILIA the "American chameleon,11 Anolis carollnensls, has many advantages as an experimental animal* Great numbers of these lizards are available In this area and may be purchased through local biological houses for a very reasonable sum* Studies in which many animals must be sacrificed are possible on this animal, whereas the cost would be prohibitive for other reptile species* In the course of 1 2
investigations upon color changes in Anolls, comparatively simple methods for many gland ablations and other operative procedures have been devised by workers who have found that It is possible to keep this animal in oaptlvity for long periods* Anolls is a member of the family of llsards known as the HKJANIDEA. Grouping and possible phytogeny of the iguanlds has been worked out by Mittleman (1942) who places the anoles In a position of early divergence from the main line of iguanid evolution* Although the anoles are by far the most common genus of lizards in the western hemisphere only two species are found in the United States* The habitat of Anolls earollnenals > as stated by Smith (1946), Is the southeastern section of the United States ranging from Tennessee to the Gulf of Mexico, and from central Texas to the Atlantic seaboard* Sex Identification In these animals Is simple* Male Individuals In contrast to the females have well developed throat fans and a pair of enlarged postanal scales* All Anolls species are small; when fully grown the male Anolls carolinensis reaches a total length of twelve to fifteen centi meters and a weight of five to six grams; the female rarely exceeds twelve centimeters and four grams* Unlike most lizards, which have a brief breeding season In the spring end lay a clutch of eggs,Anolls carolinensis lays single eggs in regular succession every two weeks throughout the breeding season,which begins In March and Is continuous through August (Greenberg 3
and liable, 1944,)* The number of animal a readily found in a particular area varies with the season and the weather* In cold weather the animals hide under dead leaves, in cracks in trees, under piles of wood, or buried in loose ground* The anole is a carnivorous animal and is attracted to its meal by the motions of its victim* Living Insects such as meal worms, flies, moths, crickets, and occasionally small roaches are accepted (McClure, 193®)» Since the animal in nature licks dew and raln-drops from leaves and twigs, one must frequently sprinkle the foliage of the cage or captive lisards will die of dehydration* The best known characteristic of this animal is its ability to change color under varying conditions of light, temperature, and excitement* After extensive Investigation on the mechanism of color control in Anolls* Klelnhola (193®) and other workers have proved that these changes are primarily under the influence of the endocrine glands* Before the animal could be used for biochemical research, a knowledge of the composition of the blood, urine, feces, and ash was thought to be necessary* Since so little is known of hibernation and seasonal changes in KEPT ILIA, a periodic Investigation of certain metabolic factors was under taken* Consequently, this paper will consist of two general sections: first, a survey analysis of blood, urine, feces, and ash of Anolls carolinensis: and, second, seasonal metabolic studies* SURVEY ANALYSES
Blood Colleotion and Handling All survey analyses on Anolls blood were done on reoently captured animals. which were fasted for four days before use* Blood for these analyses was obtained in the following man ner* The plunger of a tuberculin syringe was thinly coated with silicone jelly and placed back into the body of the syringe* About 0*001 ml* of a 0.004 pbr cent solution of heparin was added to the tip of the syringe, drawn into the chamber and spread over its wall* Silicone jelly was placed on the syringe adapter and a number 26 needle was fitted on the syringe. Through such a procedure a practically airtight syringe was obtained* While the animal was held ventral sur face up on a work table by an assistant, the needle of the syringe was placed just anterior to the pectoral girdle in the midline, parallel to the body of the animal and pointed toward its tail. The needle was then carefully pushed through the skin into the internal region just beneath th© ventral surface of the pectoral girdle* With careful probing the tiny heart (0*3 - 0 *6 cm.) was penetrated, and following slight suction on the syringe, blood was obtained. The needle was allowed to remain in position for a short period A 5 after the fir at evidence that the heart had bean entered in order to allow the organ to refill* From a single animal one can obtain a maximum of 0*05 to 0*20 ml* of blood* If less than half of the total estimated blood volume is drawn* seven out of ten animals will survive such a blood letting* If blood pH was to be determined or If whole blood was required* the pH capillary tube and the blood pipettes were immediately filled directly from the blood In the syringe. If plasma was required* a metal clamp was fitted to the syringe between the head of the plunger and the body of the syringe and the blood was then centrifuged In the syringe (Figure 1}» If acid base studies were to be done* a drop of mineral oil was layered above the blood* After centrifuging the syringe was held vertically and the plunger carefully twisted upward* By this technique practically all of the
0 .0 3 to 0*15 ml* of plasma was measured directly into blood pipettes for analyses without disturbing the packed cell layer in the syringe* 2• General Blood Studies An investigation of the erythrocyte count, packed cell volume and oxygen capacity of the blood of Anolls was under** taken in the month of November* The capillary hematocrit method of van Allen (192$) was used to determine packed ©ell volume; a Spencer *Bright-linen counting chamber was used In the determination of cell count (Todd, et al, 194B)s and oxygen capacity was measured by a modification of the 6
I No. 26 Needle Syringe Adapter A Syringe Body - - No. 7 Cork —
-Mineral Oil------tf- Blood------
Syringe Plunger Tube holder of a Clinical Centrifuge------
------Metal Clamp------
Head of Plunger febz'sL Figure h BLOOD CENTRIFUGING 7 syringe-analyser method devised by Houghton and Soholander (1943) tor determining oxygen content, The capillary pipettes for the cell count and oxygen uptake determinations as well as the capillary hematocrit tubes were filled directly from the syringe* The results of this study are shown in Table I* The average value for the red cell count was close to that obtained by Habalais (1936)* For comparative purposes a compilation of similar work on various lizards is shown In Appendix I* TABLE I Erythrocyte Studies
Average Mode Maximum Minimum Packed Cell Vol. 28 29 34 20 26 <*> Red Blood Cells 0 *9 6 0*97 1 .2 1 0.61 26 (10® per mm*3) Oxygen Capacity 9.3 6 *6 1 2 .5 7.0 15 (Vol. %)
3 . DeterminatIon of Blood and Plasma Electrolytes Techniques were devised so that determinations of pH,* Na»
K, Cl, and CO2 could be done on the plasma from one animal* Analyses of sodium and potassium In plasma and whole blood were made with the Beckman flame spectrophotometer. Chlorides were analyzed in plasma and in protein free filtrates of whole blood with the method of Schales and Schales (1 9 41) adapted to smaller volumes of sample through the use of the ultramicro burette of Oilmont (19 46) and techniques of ultramlcroanalysls a suggested by Kirk (195 O)* Carbon dioxide in plasma and In whole blood was done by the syringe-analyzer method of Scholander and Houghton (1943)* The Beckman model Q pH 1 meter, fitted with the Beckman capillary glass electrode, was used to determine blood pH* For Ha-K analysis 0*02 ml* of plasma or whole blood was added to a 10 ml* volumetric flask* The flask was filled to the mark with water which contained 0*04 per cent Sfcerox SE , 2 a surface tension lowering agent* The unknown solution was then passed through the atomiser and flame of the flame spectrophotometer and the emission intensities at 589 milli- micra (Na' and at 767 millimlcra (K.) were bracketed between standard solutions. These standards contained sodium and potassium in very nearly the same ratio of concentrations as were present in the unknown in order to rule out the effect of mutual excitation* Determinations of calcium and. phosphorus in the plasma were done on single samples of blood from the larger animals* Calcium was done on 0*08 to 0*10 ml* of sample using the boric acid titrimetric method of Sobel and Sobel (1939) end the
Gilmont ultramicroburett* Phosphorus was done 00lorimetrieally in the microtubes of the Klett^Summerson colorimeter by the method of Kuttner and Cohen (1927) adapted to 0*05 ml* of plasma. Table II summarizes the results of the electrolyte studies on the blood and plasma*
Beckman Ho. 290-63 2 Monsanto Chemical Co. 9
TABLE II Blood Electrolytes (millimoles/liter)
Average Mode Maximum Minimum Analyses Month PLASMA pH 7.26 7 .1 6 7.63 6.93 25 001 . H& 157 159 186 139 41 Oet« K 4.59 4*48 5.93 2.80 15 Cot. Cl 127 128 133 113 30 Oct. COo 15.4 14.5 22.5 9 *6 25 Cot. Ca 2.9 3.1 4 .2 2 .0 20 Aug. P 2 .6 2.7 3.2 1.7 19 Aug. BLOOD Ha 130 125 135 118 15 May E 3 1 .8 33.2 38 .4 20.9 15 May Cl 105 107 116 78 18 May C02 1 4 .0 1 3 .8 17.1 9.3 15 May 10
4* Blood and Plasma Nitrogen Studies Tungstic and trichloracetic a©Id precipitate® were used to determine blood and plasma protein* Thea© precipitates vere digested with the digestion mixture suggested by Kirk (1950)* Fractions of the clear digest vere added to Conway diffusion cells and the protein was determined as NH3 {Conway* 1940)* Urea and ammonia were also analysed In the Conway cells (Conway, I94O), The method of Brown (194?) was used for the analysis of uric acid* Hesuits of nitrogen studies on blood are shown in Table XXX•
Table XXX Blood nitrogenous Constituents
Average Mode 1 wtiiB MinImUm Analyses Month Blood Protein 11.5 1 0 .0 13*6 9*1 16 Apr. (?) Plasma Proteins 4.1 3*9 3*1 3*0 12 Apr. {%) Blood Uric Acid 7*9 6.5 1 0 .a 4*1 20 Apr. (mg.#) Blood Urea 7*2 7.9 9*6 3*5 15 •Tan. ( mg •#) Blood SHU Tr. 0*9 0 1$ Apr. (mg.#)
5 . Blood Sugar Some 71? determinations of blood reducing substances were done using a micro modification of the method of Pol in and Wu (1920)* This method like other procedures for blood glucose is not spec If io but is actually a measure of the reducing substances of the blood* To determine in a more specific manner that fraction of the blood reducing substance 11 attributable to glucose, a series of f©mientation studies vere carried out using a yeast microdiffusion method in a special Conway cell (O'Malley, efc al, 1943). When possi ble, a parallel determination was done by the Folin-Wu method. The results of these Investigations are indicated in Table IV* Henceforth in this paper the total reducing substances of the blood as measured by the Folln^Wu method will be described as blood sugar or blood glucose* TABLE IV Comparison of the Hesults of Two Methods of Determining the Reducing Substances In the Blood (All values are expressed as mg. % glucose)
Animal Ho. Fermentation Bolin and Wu 1 190 200 2 194 3 160 180 4 162 5 1-49 6 164 167 7 141 184 8 173 9 141 203 10 lie 184
Average 159 186
There are seasonal variations In the blood glucose, but these will be discussed at length in the section on metabo lism. The average glucose over the year Is 1?2 mg. per cent and there are no sex differences In these values* Early in the sugar studies It was found that blood from decapitated animals gave the same values as blood obtained by the more 12 tedious cardiac puncture. As these same animals were to be sacrificed for other studies, all glucose determinations except the early studies were done on blood obtained by decapitation. Since the blood sugar was comparatively high, the possibility of an emotional hyperglycemia had to be ruled out. A glucose level of 16? mg. per cent was obtained on a group of 20 animals treated with extreme care and decapitated immediately upon removal from their cage. Twenty other animals treated without special precaution had & blood glu cose level of 174 mg. per cent. Anolls apparently does not exhibit the marked emotional hyperglycemia as was shown to occur in the horned toad Phrynosoma (Kedfield, 1918)* 6. Urinary Constituents All urine analyses were done in October on animals which had fasted four days. Sample collection is a simple task. The animal is turned ventral surface upward and slight pressure Is placed on the abdomen Just anterior to the anus. As the drops of urine appear at the cloacal orifice, they are drawn by capillary action into a blood pipette which rests upon the work table. With such a method as much as 0.08 ml. of urine may be collected from a single animal, but on the average about 0.02 ml. is obtained per individual. Unfortunately, It is difficult to collect twenty-four hour urine samples from these animals. Therefore all urine values are expressed in weight-volum© unlta rather than as 13 weight per twenty-four hour sample* Urinary pH, Ha, K, CQg, HBj, and urea were done toy the same techniques used in blood analyses* In the pH deter minations the drop of urine was directed from the cloaca! orifice into the capillary electrode* The method of Folin (1931) was used to determine uric acid* Due to the high concentration of uric acid in the lisard urine, a greater dilution of the original sample was required than that specified in the published method* The method of Folin and Wu (1919) was used for creatinine analyses* The experimental results are shown In Tables V and VI* The sum of urinary oations determined is considerably higher than the sum of anions* This may be due to a high concentration of sulfates, phosphates or organic acids in the urine*
TABLE V Urine Electrolytes (mllllmoleo/llter)
Average Mode Maximum Minimum Analyses pH 5*51 5-36 6 .5 0 1*62 19 Ha 20*2 20*6 3 3 .7 5*7 1a R 17.1 15*1 3 6 .9 5.1 16 Cl 23*1 21.6 4.9 .0 1*0 22 C02 Tr* 1.0 0*0 15 m z 5 .8 1*1 16 .3 3 .5 20 u
TABLE VI Ur In© Nitrogenous Constituents (nig. %)
Average Mode Maximum Minimum Uric Acid H 45*6 3® *3 166.5 5*3 19 Urea N 6.6 10.2 16.5 4*9 20 Ammonia N 6.2 6.2 22.6 4*9 20 Creatinine N 1.3 1.0 1.9 0.6 16
7. Fecal Analysla Fecal samples were collected on a layer of non-absorbent paper placed overnight on the floor of the animal cage. The droppings obtained were dried, ashed, and analyzed for mineral constituents* The methods used in fecal ash analysis will be discussed In the section on animal ash analysla. The droppings collected by the above method unavoidably include Urinary constituents. Two uric acid analyses were made on droppings using the method of Folin (1934). Uric acid amounted to 45*0 and 63*3 per cent of the dry weight of these samples. Chitin la not digested in any appreciable quantity, If at all, by the anole. One can easily recognize In the droppings undigested chltlnous skins of the meal worms of the diet. Table VII summarizes results of the analysis of two samples of droppings. TABLE VIX J Fecal Analyses (mg.)
....IKT .. Insoluble Dry Wt. Ash Wt. Residue JL 0 a 01 Sample 1 542 62.5 20.4 0 .4 0 1 *33 2.57 0.43
Sample 2 1,854 143-0 84 • x 2 .8 5 4*65 1 0 .3 2 2 *34
8 • Composition of the Animal Ash Ash studies were carried out in November*1951, on animals recently caught* After a four day fast they were weighed, decapitated, dried at 75 ° 0 * to constant weight, and ashed in a muffle furnace at 450-500° C. The resulting ash was weighed, ground to a very fine powder, placed in a
10 ml. volumetric flask and diluted to 10 ml. with distilled water. The supernatant fluid was analysed then for chloride, sodium and potassium. Following the analyses of these water soluble constituents, enough HC1 was added to the flask to dissolve the water insoluble calcium and magnesium salts. Water was added to the 10 ml. mark said samples of the acid solution were analyzed for oalcium and magnesium* Ash Ha and K analyses were made using the same technique described under blood analyses. The micro diffusion method of Conway (1940) was used to determine ash chloride. Calcium In the HC1 solution of the ash was precipitated with ammonium ox alate and determined titrimetrically with permanganate (Kolthnff and Sandell, 1936). A sample of the supernatant solution 16 after precipitation of calcium oxalate, was analysed for mag nesium by an adaptation of the colorimetric method of Denis (1922). The results of the ash analyses are shown in Table VIII* 9• Discussion and Analysis of Data The distribution of body water in Anolls may be com puted with the use of the data presented in this chapter and the following assumptionst First, the ash chloride originates from the extracellular fluid only $ 3 second, the chloride con centration of the extracellular fluid is the same as that of the plasma;^ and finally, the blood volume of Anolls is 3*02 per cent of its body weight, the value found by Fry and Adelaide (1914) for the lizard, Lacerta agilia {Figure 2)* Jsing the averages of the five ash determinations, the hematocrit value of 28 per cent, and the plasma chloride average value of 127 meq./liter, the distribution of water in the average 3 *34- gram animal is then* Total Body Water 2*25 g • Intracellular water 1.-49 g. Extracellular water 0*76 g. Interstitial water O.69 g* Plasma water 0*07 g# Blood and urine studies upon Anolls indicate that this animal is more acidic than the turtle and the alligator*
Both the ph and CO2 content of the blood and urine of Anolla
3 The red blood cello of Anolls contain 15 mllli- eqaivalents of chloride per liter of ceils.
^ The ratio Na/Cl averages I.24. in the plasma and 1*23 in the aah* TABLE VIII Ash Analyses on Specimens of Anolls carollnenals (Kov* 1951)
Wet Dry Ash Sex Wt* Wt* Wt. Na Cm Cl L | Ash. Ha K Ca Cl (g>) (mg.) bod; wt.) {% ash wt.) ■. 3.50 1.32 0.175 3.35 5.87 58.3 1*34 3*85 62.3 5.0 1.91 3*35 33*3 0.77 2.20 H. 3.26 0.99 0.135 8.86 5.23 43*2 1.10 2.73 69*6 4.1 2.12 3*87 3&*2 0.82 2.03
K. 3.02 0.90 0.132 2.23 5*03 4 5 .6 1.19 3*30 70.2 4.4 1w69 3 .8 1 3 4 .6 0 .9 0 2 .5 0
H. 3.71 1.26 0.173 2.39 6.23 57.9 1.30 4*20 66.0 4*7 1.38 3*60 33.5 O .75 2.43 P. 3.22 1.00 0.153 2.80 5.65 51.6 1.39 3.12 68.9 4.8 1.83 3.70 33.7 0.91 2.04 *3 .3 4 1*09 0.155 2.73 5 .6 0 51.3 1*26 3.44 67.4 4*6 1.76 3 .6 1 33.1 0 .8 1 2 .2 2 ZA %_ P lasm ajh2Ql_ __ 20.6 % INTERSTITIAL H20
44.7 %
INTRACELLULAR H20
32.6 % SOLID
Figure 2 WATER DISTRIBUTION 19 and ot&er lizards are low when compared to values found in the turtle and to a lesser extent In the alligator* The chloride ion of the blood is also much higher in the lisards and snakes than in the other orders of RE PI ILIA {Appendix II)* In some of the earlier acid-base analyses on Anolia» extremely low values of blood CO^ (3 meq#/llter) and pH (6*70) were found. These values were not included in the preceding tables because the analytical techniques at the time were thought to be untrustworthy. If there 1® any validity to these low values» they may Indicate that the anole resembles the lizard, Bauromalug» in responding to high temperatures with an increase in acidity and a reduction in available base since these questionable values were obtained in August (Dill, et al, 1935)# A high plasma inorganic phosphorus seems characteristic of lizard species* This fact plus the apparent great variability In blood pH and plasma Ca found In the REFT ILIA recommend these animals as research tools for Ca-P studies* Blood sugar In the lizards Is higher than that found In the blood of members of the other reptilian orders (Appendix 111)* The values for the lizard compare closely with blood sugar concentrations found In some of the birds (Dyer and Hoe, 1934). Perhaps these high blood sugar values are associated with the fact that the lizards as a group have a higher standard metabolism than do the other reptilian orders (Benedict, 19 32). 20
A study of the ion concentrating power of the Anolia kidney was made by studying the ratio of urine concentration to plasma concentration of electrolytes analysed in both fluids. The values found indicate that the ancle kidney has a low coneentrating power for sodium and chloride but concen trates potassium 3 *70 times* Hence, the kidney tends to retain sodium and chloride and remove potassium when the animal Is on a diet of meal worms* METABOLIC STUDIES
I* Introduction Throughout 1951 analyses were dona each month on Anolia to compare the effect of the seasons on animals obtained fresh from the wild with groups kept under laboratory conditions* In order to determine whether seasonal temperature changes are primarily responsible for the metabolic variations found in the animal, a colony of captive lizards was main* talned at the average summer temperature of Hew Orleans, 26° C« Because of the importance of light In the behavior responses of the anole, the hours of light to which the captive animals were exposed were^aried* The captive colony was divided into two groups* Since south Louisiana receives fourteen hours of sunlight in June and ten hours in December, one group was exposed to fourteen hours of light and the other group to ten hours of light each day* The light and dark colonies were kept In separate screen wire cages whose dimensions were 2*5 by 3*0 by 1*5 feet* The cages were placed in a constant temperature room beneath a battery of four, forty watt, white fluorescent lamps* An automatic clock control was set to turn the lights on at 21 22
8 A* M. and off at 10 P. M. To obtain an additional four hours of darkness for the dark adapted group, a black cloth
was placed over this group from 8 A- M« to noon of each day. All animals that were eventually used in the light and. dark ex periments had been exposed to the fluorescent light of their respective cages for at least three weeks* By the end of three months the colony was totally depleted by the frequent sacrifice of the animals for analyses and consequently required restocking* Every four days the colony was fed meal worms, the larval form of the Insect, Tenebrjo, which Is composed of about 2 per cent glycogen and 1$ per cent fat (Wiggle a worth, 1950)* Since Anolis will not drink from a dish but acquires its water from drops on the leaves of plants, water hyacinths, ivy and ferns were kept in the cages during the investigation and this foliage was sprinkled generously once or twice each day with fresh water* In all of the metabolic analyses, the three experimental groups were used. Henceforth in this paper these groups will be identified as: a* Nature Group-animals obtained each month directly from the wild. b. Light Group-captive animals which had been kept at 28° C* and exposed to 1 £ hours of light per day. c. Lark Group-captive animals which had been kept at 28° 0 . and exposed to 10 hours of light per day. 2* Analytical Routine Fifty animals were sacrificed from each of the experimental 23 groups each month. Twenty of these animals were used for blood glucose and liver studies, twenty for analyses of total lipid and ten for respiration determinations * All animals were fasted four days prior to use since it was found that all traces of food disappear from the digestive tract within this period. The animals used for glucose and liver studies were weighed, sexed, decapitated and 0 *0 $ to Q .10 ml. of blood wsb taken up in a blood pipette and added to the tungstlc acid solution in a centrifuge tube. The animal c&rcasa was opened quickly down the midline and the liver removed by cutting the hepatic vessels and a piece of con nective tissue between the right lobe of the liver and the right gonad. The liver was weighed quickly on a torsion balance and dropped into boiling 30 per cent RGH solution. Glucose was determined on the tungstlc acid filtrate and glycogen was done on the alkaline liver hydro lysate by the method of Good, et al (1933)» Animals used for total lipid analysis were sexed, weighed, killed and dried to constant weight in a vacuum desiccator with concentrated HgSO^ as the deslecant. After the dry weights were determined, the animals were ground Into fine pieces and extracted with ether. The ether extract was filtered, evaporated and the lipid was weighed. Respiration was studied in a closed space respiration chamber (Figure 3) which consisted of a liter ©rlenmeyer flask fiowse 3i n 25 fitted by means of a three holed rubber stopper to a manometer thermometer, and a pinoh clamp for pressure adjustment, The animal was suspended from the bottom of the rubber stopper of the re spirometer in a small cotton bag (5 by 12 cm.} made from light net cloth such as is used in making curtains The carbon dioxide produced by the animal was absorbed by a standard Ba( OE}^ solution which covered the bottom of the flask* Oxygen utilisation was measured by the decrease in pressure in the flask as indicated by the manometer. After the animals were sexed and weighed, they were placed into the respiration sacks and conditioned In a darkened incubator at 26° C* for four hours. The animal and sack were then tied to the manometer unit, and the entire unit was stoppered into the erlenmeyer flask which contained 25 ml. of standard Ba(01)^ solution. The respirometer was placed into the darkened Incubator. After an hour the height of the fluid in the manometer was leveled and readings of time, temperature and atmospheric pressure were taken. Forty-four to fifty-two hours later readings of these same variables were taken, the manometer level was measured, and the Ba(OH) was titrated with standard HC1. Oxygen utilisation A was calculated from the pressure, temperature and manometrlc
^The position of the animal In the respiration sack mimics to a certain extent the tendency of the chameleon in the wild and In captivity to rest between surfaces* 26 changeb and carbon dioxide production from the result® of the titration* 3• Seasonal Glucose and Liver Studies Blood sugar values vary with the season. In the late winter and early spring the blood of Anolis contains nearly 200 mg. per cent sugar. This value drops to a minimum of about 100 mg. per cent in late summer. All experimental groups had practically the same average blood sugar values and exhibited the same seasonal periodicity. Tables IX and X summarize the results of the blood sugar studies. Liver glycogen averages about one per cent of the wet liver weight from March until September. In the fall the glycogen content of the liver Increases to an average of about two per cent of the wet liver weight. As the liver weight in proportion to body weight also increases In the fall, a table of liver glycogen as per cent body weight presents a more accurate picture of this Increased seasonal glycogen storage (Table XI). Although all experimental groups exhibited the same periodicity In liver glycogen, the livers of the captive animals, probably due to better feeding, were always heavier and contained more glycogen than animals from the wild (Table XII). Figure 4 shows a composite curve of the liver weight of the captive animals expressed as per cent body weight plotted on a chart adjacent to the curve of the same values of the Nature Group. A series of peaks Is evident on the chart of ABLE IX Seasonal Blood Glucose (mg. %)
Month Total Nature Light Dark Average 1951-52 Analyses Group Group Group All Groups
Jan. 18 215 21? 216 Feb. 39 213 205 210 March 54 206 214 186 202 April 60 237 190 176 206 May 55 204 177 176 187 June 48 153 173 200 177 July 51 186 211 211 178 Aug. 60 103 122 68 112 Sept. 80 138 160 16? 155 Oct. 55 163 151 158 157 Nov. 55 189 135 108 146 Dec. 60 146 149 148 Jan. 30 173 173 Feb. 30 168 201 184
TABLE X Group Blood Glucose (axg. ft)
Group Analyses Average Nature 231 172.5 Light 230 174.2 Dark 254 168.9 Total 715 171.7
WWW aa
TABLE XI Seasonal Liver Studies
Month % Liver Liver as Liver Glycogen as 1951-52 Determinations Glycogen % Body Wt* % Body Wt.X 10* 2
Jan* 39 1*51 2 *6 4*23 Feb. 36 1*00 2 »9 2*90 March 40 1.19 2*9 3.45 April Aa 0.99 2 .1 2,08 May 59 0*96 2*7 2*59 June 49 1*06 2 *9 3.07 July 52 1.07 2.7 2*89 AUg* 56 0*84 2*7 2.27 Sept* 37 2 *1 1 4*2 8.65 Oct* 41 1*79 4 .0 7 .6 6 Nov* 54 1*23 4*3 5.37 Dee* 30 1 .6? 3.4 6 *36 „ Jan* 15 1 .0 0 3 .0 3 *0 0 Feb* 14 1*70 3.0 5.10
* Includes only animals from the Nature Group
TABLE XII Group Liver Studies
Group Da tormina t lonei % Liver Glycogen Liver as % Body Wt Nature IBS 1*17 2*53 Light 193 1*26 3*29 Dark 191 1*50 3*50 °• / o
1 FEB. APR. JUNE AUG. OCT. DEC. FEB.
Figure 4 : Liver Weight as % Body Weight
— Captive Animals Nature Group I Date Colony Restocked ?o s O 30 the captive animals which la probably an art If act caused by the periodic replenishment of the supply of captive animals» The arrows above the curves indicate the dates on which the new groups of animals were placed in captivity* In order to test the possibility of keeping the weight of the large livers of the fall animals up by feeding, a group of llaards was placed under dark colony conditions In November and their livers were analysed in February* Although these animals ate well in the season when wild chameleons have a scarcity of food* the livers of the Dark Group decreased in weight in the same manner as the group obtained from nature* Some 700 determinations of liver weight were done throughout the year* A distribution graph of these values for the Hature Group was plotted and was found on analysis to present two series of points; the January through August weighings were evenly arranged about one series whereas the values of weighings from September to December were In another series (Figure 5)* Seasonal Lipid Studies The ether extractable lipid of the dried animal carcass reaches a very low value in late spring and then Increases progressively, reaching a maximum in the month of November after which it begins a gradual decline (Table XIII). Animals kept in captivity apparently do not store fat to any considerable extent* The average lipid of the captive LIVER WEIGHT (mg.) 250 200 150 100 iue : ie Wih pr nt oy egt Ntr Group) (Nature Weight Body unit per Weight Liver 5: Figure 50 2 3 T H G I E W Y D O B ° / \° 4 5 (g.) 6 7 8 vo H 32
TABLE XIII Seasonal Lipid. Studies
Month Total Lipid as Water^as % Lipid 1951-5& Determinations % Body Wt. free Body wt.
Jan* 40 2.0 74.7 Feb. 39 2.3 75.9 March 43 1.9 75.5 April 33 1.0 76.3 May 30 0.8 74.6 June 34 4.5 74.2 July 36 2.0 73.8 Aug. 48 2.>7 75.4 Oct. 56 3.5 74.4 Kov. 86 5.4 73.2 Dec • 60 4.0 70.9 Jan. 30 4.5 73.8 Feb. 14 4.0 72.2
TABLE XIV Group Lipid Studies
Total Lipid as Water as % Lipid Group Pet ermine t Ions % Body Wt. Free Body Wt. Mature 176 3*13 74*4 Light 165 M*91 73*7 Dark 12S £iZ2 Total 561 2.93 74-2 33 animals mas found to be slightly lower than that of the wild animals (Table XIV). In August Park Group animals which had been In captivity for three months were a pale blue-green color when in th© melanophore contracted state rather than the bright yellow- green color of the wild chameleon* Th© lipid extract of this Park Group was found to be less yellow in color than were the extracts from th© Light Group and th© Mature Group* The absorption spectra of th© yellow pigment was determined somewhat crudely by the us© of th© Xlett-Summerson photoelec tric colorimeter* Since the position of the absorption maxima and minima corresponded closely to that recorded for heta-carotene, the concentration of these yellow lipids was estimated by the use of the colorimeter with a 420 millimicra filter. The results were expressed in terns of an artificial color standard of KgCrO^, an inorganic compound of similar color (Table XV). TABLE XV Pigment Studies (All values are expressed as g. X 1G~5 of K^GrO^)
Nature Light Dark Weeks Light and Dark Date Group Group Group Groups Captive 23 Aug. 173 66 26 12 24 Oct. 173 135 67 3 27 Deo. 236 181 6 4 Jan. 190
Although the llpld of the Dark Group contained th© least quantity of yellow pigment, th© lipid of both captive groups M contained less pigment than that of the animals from the wild. The cause of these variations has not been critically investi gated) however, the lipid extract of the meal worm is very low in yellow pigment and this lack of carotenes In the diet is probably responsible in part for the low pigment content of captive animals* Nevertheless, the distribution and chemistry of the carotenes in Anolia warrants further study* 5* Studies on Lipid and Glycogen Distribution Due to the marked seasonal changes In the liver, a more complete series of liver analyses was begun in November* The bar graphs of Figure 6 depict th© results of these analyses. Liver lipid content decreases progressively from th© high of 28.9 per cent in November to 9*4 per cent in February) liver glycogen during the same period falls from 1 .6 per cent to 1.0 per cent* The charts of the livers of the captive groups show little change in total lipid although the total glycogen content of the livers of these animals rises over the same period. The results of th© Dark Group analyses performed during the months of December and February are especially interesting. Th© animals were placed in captivity in late November when their liver weight as per cent body weight was very high. Although these animals at© well their liver weight decreased without any marked change in liver composition* A series of studies was also done on the distribution of glycogen and lipids in the liver and in the hepatectomised carcass* During the winter months the store of glycogen and % % LIVER W E I G H T 100 75 50 25 iue : IE ANALYSIS LIVER Figure 6: 21 aue Group Nature 68 Nov. Water a. 14 5 Jan. mi Feb. 3 0 Nov. 27 Dec. 27 0 Nov. 3 Feb. ih Group Light Lipid Nv 27 e. 14 Feb. Dec. 7 2 0 Nov. 3 ak Group Dark Glycogen lipid built up in th© autumn is slowly depleted* Th© rat© of depletion of glycogen is much more rapid in the carcass than in the liver (Table XVI). The lipid of th© liver, on th© other hand, is depleted more rapidly than that of the carcass (Table XVII)in the Nature Group. TABLE XVI Distribution of Glycogen in the Liver and in the Bepateeternised Carcass (mg./lQO g. Body Weight)
Nature Group Dark Group Date Liver ffareasa Total Liver Carcass Total 24 Sept. 51 229 260 125 239 364 14 Feb. 23 63 66 73 39 112
6. Seasonal Respiration Studies There are no marked seasonal variations In the oxygen utilization or carbon dioxide production in Anolis. All changes from the mean are of the order of 10 to 15 per cent and are sussaiarlzed in Tables XVIII and XXX. From March through June oxygen utilization measured at 28° C. averages
0 .1 9 8 ml./ g. body weight/ hour, whereas during th© remainder of th© year it averages 0 .2 2 0 ml./ g* body weight/ hour. Although the mean R. Q. is high in all seasons, the lowest individual values recorded occurred in April in animals from the Nature Group. The highest mean recorded was for the period from November through February, the season of high glycogen storage and elevated glucose. TABLE XVII Distribution of Lipid in the Liver and In the Hepatectomlaed Carcass (g./lOC g. Body Wt.)
Nature Group Light Group Dark Group Date Liver Carcass Total Liver Carcaea Total Liver Care as a Total
25 Nov. 1.06 3-31 4*37 0,45 4*73 5*1$ 0.50 3*62 $.12
27 Dec. 0.41 2.93 3*34 0*53 4*06 4*59
5 Jan. 0.60 3*93 4*53
14 Feb. 0.26 3*96 4*24 0*59 3*27 3*66 TABLE XVIII Oxygen Consumption of Anolla c&rollnensls at 28° C. (ml. of 027g. Body Wi;/hr.)—
Determinations Average Mean Error* Nov. through Feb* 219 0*220 0.004 0*065 March through June 183 0.198 0.004 0.052 July through Oct. 206 0.223 0.004 0 *0^(9 Nature Group 180 0.227 0.004 0.056 Light Group 210 0.211 0.004 0.053 Dark Group 216 0.206 0*003 0.051 Males 462 0.213 0.005 0;055 Females 146 0.222 0.005 0 .0 5 7 Total 606 0.215 0.002 0 .0 5 4
TABLE XIX Respiratory Quotient of Anolis earolinenals at 28° C. (vol. 6if Sg/vol. 1S3^)
Determinations Average Mean Error*3* A* Nov. through Feb. 210 0.929 0.006 0 .1 2 0 March through June 169 0.910 0*009 0 .1 1 9 July through Oct. 198 0.902 0.007 0 .103 Nature Group 172 0.898 0.010 0 .1 2 4 Light Group 204 0 .9 2 0 0.008 0.113 Dark Group 201 0.922 0.009 0 .1 25
Total 577 0.914 0 .0 05 0 .1 1 4
fiSequals the mean deviation/determination Jj£d/n-l where d equals the deviation from the mean of a determination and n equals the number of determinations* Th© mean error equals (H/• 39
The yearly average of 0,215 ml./g*/hr* oxygen consumption at 2i° C* Is much lower than the 1*034 ml*/g*/hr* at JO0 0* obtained by Clausen and Mofshin (1939) In * study of th® photoreceptors of Anollc* These workers were not Interested In standard metabolism and as a result their values were obtained from determinations of only three hours duration and on animals poorly adapted to the respirometer* IXi® to the many variables which were inadequately controlled* their values must be far removed irom the true standard metabolism of the anole • Benedict (1932) showed that the metabolism of the python at 26° C. Is 200 to 600 per cent above the fasting level after a protein meal* In order to check the effect of feeding on Anolls. oxygen consumption and carbon dioxide production was determined on twenty animals after a heavy feeding of meal worms* The results indicated that the metabolism of the chameleon on such a diet Is only 10 to 13 per cent above the fasting level* This Increase occurs on the first two days following feeding*
7 # Summary of Seasonal Metabolism (Figure 7) In the late summer and fall the body weight and to a greater extent the liver weight increase due to the storage of glycogen end fat. With the increased storage of liver glycogen the blood glucose falls to a low level* By November the lipid and carbohydrate content of the body represent close to $5 kilocalories per 100 g. body weight* Only 3 per cent of this reserve 4 0
BLOOD GLUCOSE TOTAL LIPID 250 (in mg. % ) (in mg. % )
200 mg. °/o / 150
100
Apr. June Aug. Oct. Dec. Feb.
.0 9 LIVER LIVER 2.0 GLYCOGEN GLYCOGEN (as % L iver Weight] (as % Body Weight) .0 8
.0 7
.0 6
Apr. June Aug. Oct. Dec. Feb. .0 5
3.5 LIVER WEIGHT (as % Body Weight) .0 4
3.0 .0 3
2.5 .0 2
2.0
Figure 7; SEASONAL VARIATIONS ANOL/S CAROLtNENStS (Nature Group) 41 energy, however, is due to carbohydrate* Even if we assume that the calorie requirement of the anole does not change during starvation, this stored energy would keep th© animal a liv e f o r 25 days at summer temperatures without touching protein reserves*
Hibernation in the lizard occurs during adverse weather conditions of the winter. Blood glucose rises and a slow decrease in stored energy occurs* On the colder days the animal buries its e lf in loose ground or hides in some other protected place. As long as th© temperature remains high and the days bright, however, the animal w ill eat although its appetite appears to be less than in other season®.
In late March reproductive activities begin to enter th© metabolic picture, and by May a ll females examined contain well developed eggs and are laying at the rate of an egg every second week (Hamlett, In press). The total lip id and carbohydrate stores at this time represent only one sixth of th© available energy content of the animal In November* Repro ductive activities begin to diminish in July and th© energy storage period of the fa ll begins* No sim ilar quantitative work on seasonal live r studies on the lizards or on other reptiles could be found in th© literature. Beginning with the work of Athanaslu (1699)* however, many workers have observed seasonal changes In
2 The caloric requirement was calculated from the oxygen consumption of 0.21 m l./g./hr. at 28° C. on the assumption that on© lite r of oxygen consumed Is equivalent to 4.8 kilocalorles. r-v' ih' - . °-.l\ i 42 energy storage 1 n the amphibians* t h e livers o f the frog are larger and have an increased energy content in the fall as compared with the other seasons of the year. Massooco (1938) has studied this problem in the amphibians t© a considerable degree. If the oxygen consumption of the reptiles is measured at the same temperature in the different seasons there are no marked changes. Anolis oarolinensia* Alligator missies* ippiensis (Hernandos and Coulaon, 1932), and Storerla dekayl. a snake, (Clausen, 1936} have seasonal differences of less than 15 per cent. In contrast to the reptiles the frog, Rana* requires 50 to 70 per cent more oxygen in the spring than in the winter (Tabulae Biologlcae* Vol. III). In the late summer and fall the histology of the pitui tary of Anolis carolinensla reveals evidence of marked storage and secretion. In the winter the gland progressively loses this stored material and by spring a different cell type predominates the histological picture (Paris, 1941)* These pituitary changes are in phase with and probably allied to the chemical changes found in Anolis throughout the year* bibliography
books
Baldwin, Ernest. An Introduetlon to Comparative Biochemistry. Third Edition. C r ’lHge s'"12 amEridgeunivor s i ty '#ri©a, 1949. Pp. I6 4 . Benedict, F. G. The Physiology of Large Reptiles with Special Refertfhce to the BeiTf Proauction o f Shake®, Tortoises, Lizards. a*H~AlYtgatora'. 1 fas h ln g to n s Carm e , 1 e Institute PublicationKo. 42$, 1932.
Conway, Edward J, M icro"diffusion Analysis and Volumetric Error. New York: van strand rCoT, T §40,'ivr' Fp•"Trl'i - 3$6*
Ditmars, Raymond L. Reptiles of the World♦ The Crogodilians, L i sards, Snakes, tu rtle s and "^orto'iseg oT*" the 'las tern and Western Hemispheres, hew RevlseOSTEIon, Jew Y o rk : MacmiTT&n 661,” 194?. Pp. v li - 3 2 1 .
E llin g e r, A. Oppenheimer1 a Handbueh der Biochemie des Menschen und He£ Tier^.H^TFter B IS C Trite"l'aH Te. Jems Gustav F is h e r , I 9IO.
FIorkin, Marcel. Biochemical Evolution. English Translation. New York: Academic Pre»s, 194$ * • 157. Heilbrunn, L. V. An Outline of General Physiology. Second Edition, Reviaed.' PhlladeTpKias W • B". ^auhders Co., 194®* Pp. v - 74®.
K ir k , Paul L. Quantitative Ultramicroanalysis. New Y orks Wiley and Sons, Inc . ,1950, f p"• v ~ 310." Eolthoff, I. M. and Sandell, E. B» Textbook of Quantitative Inorganic Analysis. New Yorks Macmillan (To. ,1936. Pp. viT"" i w ?— Krogh, A u g u s t. The Respiratory Exchange of Animals and Man. Monographs on Bio cnemi a try. London s Eongmans, Green and Co., 1916. 43 u
Prosser, C* Ladd. Comparative Animal Physiology. Philadelphia? W. B, SaundersnCJ3T7T95TT7
Smith, Hobart M. Handbook of Lizards* Lizards of the United States and o f Canada* Tfhaba, Ufew York $' domst o ck" ¥uEITshing co,*T",i9TET Y p * ~ w r r
Tabulae B lo lo g le a e* B e r lin * W* Junk, 1926, V o l. III*
Todd, James Campbell, Sanford, Arthur H. and St11w ell, Georg© Giles. C linical Diagnosis by Laboratory Methods* Eleventh Edition. Pliii&delpLia, £©nn. 5 f 1 * ‘B**’’ la u n d e rs C'oT, 194$® Pp. 1 9 4.
Wigglesworth, V. B. The Principles of Insect Physiology* Revised E d it io n . ''"Hew Yorks E. B* Duff on, I'ffO* BIBLIOGRAPHY Periodicals
Andreen-Svedberg, Andrea* “On the distribution of sugar between plasma and corpuscles in animal and human blood," Skandlnav i ache a Archiv fur Phyaiologie. LXVI ( 1 9 3 3 ),
Athanasiu, J. “Ueber den Gehalt des Froshkorpers an Glycogen in den verschledenen Jahresseiten,® Archiv fur Physiologle von Pfluger, LXXIV (1699), 561-$6f. “ ~ “Ueber den Hesplrationsweehsel des Frosches In den verschledenen Jahreszelten,* Archiv fur Physiologic von Ffluger, LXXIX (190G), 4OQ-42T: Austin, J• Harold, and Sunderman, F. William, and Cam&ck, J. G. "The electrolyte composition and the pH of serum of a poikllothermous animal at different temperatures,* Journal of Biological Chemistry, LXXIX (1927)* 677-665. Balli, Antonio Di. "Osservaaionl sul rapporto tra 11 peso del fegato ed 11 peso eorporeo in hacerta muralls. Laur.,® Archivio dl Science Blologiche (Naples), XXIV (1936), 5i7-52£ • Britton, S. W., and Kline, H. F. “Emotional hyperglycemia and hyperthermia in tropical mammals and reptiles,11 American Journal of Physiology, CXXV (1939), 730-734* Brown, Herman. “The determination of uric acid in human blood.® Journal of Biological Chemistry, CLVIII (1945), 601-608. Capper, Norman Steward, and McKibbin, Isabel Mary Wilson, and Prentice, James Harris. “The conversion of carotene into vitamin A by fowl,® Biochemical Journal, XXV (1931), 265-274* Carmichael, E. B., and Petcher, P. W. “Constituents of the blood of the hibernating and normal rattlesnake,® Journal of Biological Chemistry, C.LXI (1945)* 693-696.
45 4 6
Chalk oft, I, L., and Entenman, 0. "The lipidea of Mood, liver, and egg yolk of the turtle,** Journal of Biological Chemistry, CLXVI (1946), 6 0 3 * 6 8 9 . ------— Clausen, H. J* "The effect of aggregation on the respiratory metabolism of the brown snake, Btorerla dekayi," Jour xml 2 L Callular and Comparative Physiology* ffJJX 1936T#iST*3$6 .i and For is, K. O# "The effect of light upon sexual activity in the llsard, Anolis carolinenals, with especial reference to the pineal body," SBaToSISaX’lTecord, LXIX (1937), 39*53. ~ * and Mofshin, B, "The pineal eye of the lisard (Anolis carolinensis), a photoreceptor as revealed by oxygen consumption studies, *Journal of Cellular and Comparative Physiology* XIV {1^77*2^4^7** Collip, J• B. "The alkali reserve of marine fish and invertebrates, 11 Journal of Biological Chemistry* XLIV (1920), 329-344. * "The alkali reserve of the blood of certain of the lower vertebrates," Journal of Biological Chemistry, XLYI (1921), 57-59. Cook, S. F. "Respiratory metabolism of certain reptiles and amphibia.* University of California Publications in zoology, b i n - j i m r ; — ------Coulscn, Roland A., Hernandez, Thomas, and Brasda, Fred <1* "Biochemical studies on the alligator," Proceedings of the Society for Experimental Biology and'MedTc"ineV^xXTTI TT? 5 0 }, 263 -SUE. Denis, W. MThe determination of magnesium in blood, plasma, end serum,* Journal of Biological Chemistry, LII (1922), 411-415. Dill, D. B. "Respiration and metabolism of a young crocodile,* Copela, I (1931), 1*3. , Edwards, H. T. "Physicochemical properties of crocodile blood (Crocodllus aoutus, Cuvier),* Journal of Biological Chemiairy, ^C^T95n','' ^15 -530. , Edwards, H. T., Bock, A* V,, Talbott, J, H, "Properties of reptilian blood. Ill, The chuckwalla (Sauromalu3 obegua, Baird)," Journal of Cellular and Comparative Physiology, VI (19337, 37*4& * Edwards, H, T. "Properties of reptilian blood. IV. ¥He alligator (Alligator mlssisslpplensls. Haudin)" Journal of Cellularr and Comparative Physiology. VI (1935), 243-2 3 4 . Drllhon, A*, and Marcoux, F* "Etude bloehimlqu® du sang at da I1 urine d fun cheloniens Testubo mauritanica," Bulletin d* l* Sool^ta* de Chtmia BloIoHouS .’H I W TEtCH. T5FWT Dyer, H* M., and Hoe, J, H. "The chemistry of the blood of normal chickens," Journal of Mutrltlon, VII (1934)• 623- 626* ~~ ~ ' ~ Edwards, H* T*, and Dill, D. B* "Properties of reptilian blood, II, The glia monster (Heloderma su spec turn, Cope),®* Journal of Cellular and 0omparative^Kyslol6Ry^''rT1¥I (1935)# Si-ji5* ~ * Brans, LLewellyn T, "The effects of pituitary implants and extracts on the genital system of the lizard," Science, LXXXI (1935), 168-469* “ Folln, Otto, "The preparation of sodium tuagstat© free from molybdate, together with a simplified process for the preparation of a correct uric acid reagent (and some comments)," Journal of Biological Chemistry, CVX (1934), 311-314* , and V¥u, fisien* "A system of blood analysis," Journal of Biological Chemistry, XXXV111 (1919)# 81-110* ______, and Wu, Hsien. "A simplified and improved ' method for determination of sugar," Journal of Biological Chemistry, XL I (1920), 367-374* " Fry, B, K •, and Adelaide, M, B* "The blood volume of cold blooded animals as determined by experiments upon frogs and lizards," Quarterly Journal of Experimental Physiology, VII (19 14), 1&5-W2 • Js Gilmont, R. "High precision ultramioroburet," Analytical Chemistry, XX (1948), 1109* Good, C. A,, Kramer, H,, and Somogyl, Michael, "The determin ation of glycogen," Journal of Biological Chemistry, G (1933)# 485-491* Greenberg, B,, and Moble, G, K, "Boclal behavior of the American chameleon (Anolis carolinenaia, Voigt)," Physiological Zoology , XVIl (1944 J» MS-439 * Hall, F. G* "The respiratory exchange of turtles," Journal of Metabolic Research, VI (1924)# 393-401* Hamlett, George W. D, "Botes on breeding and reproduction in Anolis," Copela, (in press)* HeIstand, William A., Stullken, D» E* "Effects of autonomic drugs on color changes In Anolis earolinensls.” Anatomical Record. LXXXVII (1943)* 4487”*------^ — * — -- —“ Hernandez, Thomas, and Coulson, Roland A, "Biochemical studies on the Iguana,” Proceedings of the Society for Experimental Biofogy andMediaIne!H t l ^ -- | » end Coulson, Roland A* ”Hibernation in the alligator,” Proceedings of the Society for Experimental Biology and MSglelSe. * Hopping, AleIta. ”Seasonal changes in the gases and sugar of the blood and the nitrogen distribution in the blood and urine of the alligator,” American Journal of Physiology, LXVI (1923), 145-163* "-- '------Houssay, B« A., and Biasotti, A, "Hlpoflsis y diabetes pancreatioa ©n los batracles y los reptile®,” Socledad Argentina d© Blologica, Buenos Aires, Revista,*7DTTIW3) * 29-33. : " * — and Biasotti, A. ”Hypophys© et diabete psncreatique chez lee batraeiens et lee reptiles,” Gompt# Bend. Soc. de Biol., CXIII (1933), 469-471. M Inaba, Riqtaro. "Tiber die Zusammensetzung des Tlerkorpers,” Archie fur Physiologic, (1911), 1-8* Iasekuts, B, and Vegh, F. "Beltrag© zur Wirkung des Insulins. III. Mittelivings Wirkung of den Gasotoffweohsel der Schildkrote,” Biochemiache Zeitaehrift, CXCIX (1928), 383-3 8 9 . Karr, Walter Kleinhols, L. B» "Studies in reptilian colour changes* IX* The pituitary and adrenal glands In the regulation of the melanophores of Anolis earolinensis." Journal of'Experi- ttgntal Biology* XTTI^rjT^" m ~ m * ------I, * 11 Studies in reptilian colour changes. Ill, Control of the light phase and behavior of isolated skin,111 Journal of Experimental Biology, XV (1936)* 492-499* » and Rahn, H* "The distribution of Intermedin; A new biological method of assay end results of tests under normal and experimental conditions," Anatomical Record, LXXVI (1940), 157-172. ' Kramer, Gustav* "Welter© uatersuehungen ftber die 0msatagrees® von Eldechsen, Inbesender© ihre Abhanglgkeit von der Temperature," Zeltschrift fur Verglelehend© Physiologic* XXII (1935)* 39-79. — ---- Krehl, L., and Soetbeer, F. "Untersuehung®n uber die Warmeokonomie der poikilothemen Wirbelthlere," Archiv fur Physiologic von Pflugar. LXXVII (1899), 611-1JSS7 Kuttner, Theodore, and Cohen, Harriet £• "Micro colorimetric studies. I. A molybdlo acid, stannous chloride reagent. The micro estimation of phosphate and calcium In pus, plasma, and spinal fluid," Journal of Biological Chemistry. LXXV (1927). 5 1 7 - 5 3 1 . ------*------* Lewis, Howard B. "Some analyses of the urine of reptiles," Science, XLVIIt (1916), 376. Luck, James Hurray, and Keeler, Leonardo. "The blood chem istry of two species of rattlesnakes, Crotalus atrox and Crotalus oregonus," Journal of Biologicalr 6hemi"s£ryr L t t l t ( 1 9 2 9 7«3-TW.------tfaasoeeo, P* "Variations saisonnieres de la compositlo &u crapaud Bufo arenarum. Hansel,n Comptes Hendes Society BlologliT CXXIX (1938), 857-8WT ' MeCay, C. M. "Phosphorus distribution, sugar, and hemoglobin In the blood of fish, ©els and turtles," Journal of Biological Chemistry, XC (1931). 497-505. ~ McClure, H* E. "Some insects accepted by the chameleon," Entomological Hews, XLIX (1938), 252-254* Marshall, E. K., Jr. "Kidney secretion In reptiles," Proe.adinga of the Society for B^er.imental BlojLogy and HedlolnA, 3(XT? TTO2) , 97T-S7T.------50 Marshall, IS. &. "The comparative physiology of the kidney in relation to theories of renal secretion*11 Physiological Reviews. XIV (1934), 133-159. — -- Maxlat Carlo* "Sul rapporto ponderale tra soma e fegato in SAURGPSIBX," Scrittl Blologicl. X (1935), 161-175. Mills, T. W. "Motes on the urine of the tortoise with special reference to uric acid, and urea," Journal of Physiology, VII (1886), 153-157.------MIttleman, M. B. "A summary of the Igu&nid genus Urosaurua," Bulletin of the Museum of Comparative Zoology* Xftl ( ) , IU3-1B1. ~™n,T Hera, M. C* D. "RIcerche chlmlche, f isieo-chimiche e morfologiche sul aangue di Testudo graeea nell*estate e durante 11 sonno invernale feoraie, InafcTEuto Zoolo&ico, Bollettino, III (1925), 71-85“: ~ “ Hoble, G* K ., Greenberg, B* 15Effects of seasons, castration and crystalline sex hormones upon the urogenital system and sexual behavior of the lizard (Anolis carolinensls),* Journal of Experimental Zoology. LXXiWitt'i (1^1') ,1 1-479* 0*MalIey, S., Conway, £. J*, and Fitagerald, 0. "Micro- diffusion methods. Blood glucose," Biochemical Journal, XXXVII (1943), 278-281* Parker, G* H., and Starratt, 8* A* "0?h© effect of heat on the color changes in the skin of Anolis carolinensls, Cuvier,* Proceedings of the American AeaSemy of Arts and Sc fences» tt ( 1 W ) 1, aIs-aTO.------— --- — Poris, Ethel G. "Studies on the endocrines of reptiles. II, Variations in the histology of the hypophysis of Anolis carolinensls, with a note on the Golgi c OnfIgu rat ion In ceils of the pars anterior and pars Intermedia,11 Anatomical Record, LXXX (1941)* 99-121 * Potter, George E., and Glass, H. Bentley. "A study of respir ation in hibernating horned lisards, Phrynoaoma cornuturn." Copela, III (1931)# 128-131. Prado, J. leal. "A glicemla normal nos ofidles," Memories do Inatituto do Butantan, But an tan Brasil. XIX (19 46), 5 9-*5^. Rabalais, Roy. "Observations on the blood of certain reptiles, pieces, molluscs, and one amphibian of the Grande Isle region," Proceedings of the Louisiana Academy of Sciences, IV (1938), 142-14®. 91 Ratseredorfer, Carla, Gordon, Albert S# and Gharipper, Barry A* The effects of thiourea on the thyroid gland and molt ing behavior of the lizard, Anolis car ©linens is,** Journal Si Experimental Zoology, CXlTTm9T77CFFTr~ "--- Redfleld, Alfred 0* 11 The physiology of the melanophorea of the horned toad Phrynosoma ,w Journal of Experimental Zoology, XXVI (l^S), 27P 3 3 3 .— ------mJLmm-- Rubner, Max, wAua dem Eeben des Ealtblufcers# XI, Tell? Amphibian und Rep til ten,” Bloohemlsohe Zeitschrlft, CXLVIII (1924), 268-30?. "------Ryerson, D* L. ®A preliminary survey of reptilian blood,11 Journal of Entomology and Zoology, XLI (1949), 4-9-55 - Seholander, P# F#, and Houghton, F. J# W. 11 Micro gasometrlo estimation of the blood gases. IV# Carbon dioxide,® Journal of Biological Chemistry, CXI* (1941), 879-884# Schales, Otto, and Schales, Selma# nA simple and accurate method for the determination of chloride in biological fluids,® Journal of Biological Chemistry, CXL (1941), 879-884#------Smith, Homer W. "The inorganic composition of the body fluids of the Chelonia,® Journal of Biological Chemistry, XXXX.II (1929), 651-661# *-- * — - * Sobel, Albert E#, and Sobel, Bernard A# RMicrosstlmatIon of calcium In serum,® Journal of Biological Chemistry, CXXXX (1939), 721-72ST Southworth, F. C» Jr., and Hedfleld, A# 0# nThe transport of gas by the blood of the turtle,** Journal of General Physiology, IX (1925), 38?-4®3* van Allen, C. M# ®An hematocrit method,® Journal of the American Medical Association, LXXXIV (1^25), 2lS?-H(S5« Vara, Harry M. ”Blood studies on fish, and turtles,*1 Journal of Biological Chemistry, CV (1934), 135-137* Vlllela, G. G#, and Prado, J • L# wFlavinas e outros plgmentos no plasma sanguines de cobras braslielras,B Revlata Brasilelra de Blolog3.a, IV (1944), 469-474* Weigmann, R# ** Jahreaaeltlich© Gnterechiede in der Erythro- cytenzahl bel Laeerta vlvlpara, J&cq*,® Zpologlscher Anzelger, CXXX TI932T, i f - l T T $2 •iley, Prank H., and Lewis, Howard B. "The distribution of nitrogen in the blood and urine of the turtle {Chryaemys p i n t a ) American Journal of Physiology. LXXX1 I T w T T T " 69^*695• Wilson, J, W, "Some physiological properties of reptilian JUljd( ^XUlar ~ CoMPayatiTe Physiology, APi KNDIX I Erythrocyte Studies on Various Lizards Pecked Cell Ked Blood Oxygen « Animal Volume / Celle „ Capacity Source — (10 P«r m J } (Vol. £) Anolis carolinenals 1*19 K&balals (19 3 8 Iguana Iguana 30.0 1.03 9*5 Hernandez and Coulson (1931) Ctenos&ura acanthura 35*0 1*03 8.0 Hernandez and Cculsoa U 9 5 D Sauromalus obesua 30*7 0*90 10.S Dill, et al (1935) Belodermir*auapeegum 30*3 0.70 11.0 Edvards and B U I (1935) CcXsonyx varlegatug O .49 8*9 Kyereen (1949) £hrynoaom& sbl&re 0*75 10.0 » mtuogSTs"' eajT— 1.10 $1*7 e goeloporus maglater 1*22 H*3 it Inserta "vlva'para '{winter} 1*67 Wei^m&nn (1932) a ■ « (other seasons} l.$6 Laeerta agills 0*94 7*6 Prosser* et al (1950) taeeria maralls 1*45 12.0 8 Anguls frakilla 1.60 15*0 n ^Calculated from hemoglobin values in some eases Blood Klootrolytoft of th« KEPT ILIA A ! SI f O o | i - 3• • • dl N3l 21 sh O n *1 HO^ON>- 8 w « 04 04 ■s 2 * QQO' H H c* sOcnO O sOO'tfN H H H 2 (V H H H H H 0* 04 O* 3 3 3 2 3 0 4 0 4 H'H f > p * ( n t o o - o* c** o* "#N 04 0 *“ 0** * 04 H C4 H O o o •• • • * * ' von O m cv o H 0* HI QtKhO t H rtM r HI Hrt H H &SS a * « 140 *# H H H tA*f 040* 34 mg-4 C h ' a m * E* * * O H hi Qs<~* * • «*■<* H 4* u m 0 H ) 4 ^ I 4 0 Q ^ Q Q Q 0 4 t f \ £ > H t f N S © O «*» Hiu CilHrtH H H H c C -**J9 O c^C4 O «A-*Q H 0\H m * * • 54 APPENDIX II (Oont.) ------PESSE — w a r n ------AnInal pH Na K Ca Mg Cl Cfc P SO* Ha X MB, 01 CQs> — iTurtlea wnt/J * - —4 Pseudemys elegans13 126 3.3 A .2 4.A 61 42 1.4 0.1 TVTfooaTil?? -- 82 36 70, 3 6 7. oonolnna15 7*50 3 9 Thalassochelyg, caretta 12 136 3.8 9.2 113 gSeloae aydaa^^ 17 Turtle(species unknown) 38 iiernandez and Coulson (1951) 1l»era (1925) 12 Dill, et al (1935) Drilhon and Hareoux (1942) 13 ^Edwards and Dill (1935) Smith (1929) ^Luek and Keeler (1929) Wilson (1939) 5 Carmichael and Petcher (1945) Souttwrorth and Redfield (192 5) ^Collip (1920) 1&Khalil (1947) ^Hopping (1923) 17Collip (1921) s Austin, Sunderman and Gamack {1927} 9Coulson, Hernandez and Brazda (1950) ID Dill and Edwards (1931) v« gO lO j o 8"? ■* * cfr o Pf o Cf" 1 o &< o & O & fS H o£ Hj 5o G0 <+ o* s S {# H* 0> P E0 Ct <3 8 »■* O Animal § <+ M S o u r c e (Values are for whole blood unless specified*) otherwise CO 00 vn 4k VjJ M H H 14*1H vn p Total Protein Organic in Substances Reptilian Blood VJt <1 4 k vn O ' 4*-. « • - *■%*«• O^H •vO 0 » H Plasma Protein -O 0" 9S I; 0 [ft5B » ct w a W «H* to to 13 ct Animal H M ) n l | J i |a«£ AJ* h* Iwl |»J vO vO £»**0 © CO tot VO to H o %0 SO oo o& ot> Source H t o ) CSV CP •©tot « ** Total Protein •s3to VO tol>- tot i * 9 * « ^ * ^ Plasma Protein © e»oo' ?o PEDX III (Cont,)APPENDIX •to GO Hemoglobin O« M3 * l»J o H* VO-js. H3 O -a * tot 0s CT © vO -to Glucose VOVO j3 H s vn o © toJ 4«** -toto> O tot h £ Amino Acid N ° * 9) *S Creatine g H » m o Creatinine Phospholipid Cholesterol Ergothionine £S CP- Jt* Va> so 0 c* p * r & £ •d m • 1 w H P H & H* O QQ ft I ct s *t m fas Animal H H 0 M ■H ! vO I fcf s H H § H* Hr *0 O *Vr p Vo o i tft to W IO ^ Source ft V* S3p *0 ^sUWHO O C* H 1 wmt o M & 18 sO *£» V*J» § e Total protoin o vk -H \JT P H \It Plasma protein • * V* *- O' APPSIIDIX Hemoglobin H JM **0 w s H* O £*4 W K> \ * i Co g *y Va) O Q& Glucose & 111 H i *• $fc KPN t.) n o (C I x0 <** H O' VO o |o ' * 0 Urea I w te I K> 0& eo * « Uriel Acid ct H V > Jp w J c* ’sO H* wVR h* Amino Acid if H ee Vdfs© I Creatine »3 ■* Creatinine Phospholipid Cholesterol Ergothionino £S? APPENDIX H I (Cont.) on Dill and Edwards (1931) Drilhon and Marcoux (1942) ^Andreen-S««‘Jiberg (1933) Z1KJialll (1947) 17'rc Cay (1931) 22Wiley and Lewis (1927) 18Vars (1934) 2^Isaekut* and T^gh (1928) 19 Nera (1925) 2^Karr and Lewis (1916) Besides the above tabulation of analyses on reptilian blood, the following special determinations have been runj fhe serum cholesterol of the snake, Ore talus horrldus, was determined by Carmiehe&l and Pete her (1945) * P’or the hibernating animal the value obtained was 146 mg* per cent whereas the active animal had a level of 241 mg. per cent* Riboflavin in high concentrations was found in the plasma of the snakes, Bathrops jararaca (165 to 288 micro grams per cent) and Eudr jaa sp* (205-to 333 mierogrsms per cent) by V ille in and Prado (1944) • Hera (1925) found that the serum protein of Teatudo graeea* a tu rtle , contained 1 per cent fib rin and 2*5 per cent albumin in the winter and 3*1 per cent albumin in the summer* Chalk off and Entenman (1946) found that the blood of the tu rtle , Ghrysemys plcta, contained 322 mg* per cent total lip id f females with active ovaries had an average value of 477 sig* pe r c e n t. VITA The candidate was born In Hew Orleans * Louisiana* on December 3 0 , 1921 * He attended the public schools of that city and graduated from Warren Baston High School« In 1944# after a year and a half as a chemical engineering student at Louisiana State University* he joined the Army Air Force and was sent through the meteorology training schools at the University of Hew Mexico and California Institute of Technology* After obtaining a professional certificate in meteorology from the California Institute of Technology* the candidate spent a year in various air bases in the United States as a weather officer* and in 1945 he waB sent to Okinawa as a weather reconnaissance officer* After spending a year overseas he returned to the United States* was discharged from the Air Force* and immediately registered at Louisiana State University* After completing a year of study at the main campus and two years as a medical student in New Orleans* he was awarded a B.S* degree and entered graduate work in the Biochemistry Department of the Medical School* 60 EXAMINATION AND THESIS REPORT Candidate: Herbert Olay Dessauer Major Field: Biochemistry Title of Thesis; The Biochemistry of Anolis carolinensjs. Approved: MajorMajoi Professor and Chairman EXAMINING COMMITTEE: Date of Examination: Ifay 7, 1952______ o o o to |rJ •to ftP# tj£ sji to to to ■fr- o vnM v-»* Urea *o *o JO *o to) Uric Acid