blETHODS F O R VOLUMETRIC DETERMINATION O F F R E S H ENDOCRINE G L S N D S C . A . SWINYARD Xedical Colle,ye of tRe State of South Carolina, Charleston TN TRODU CTI ON Many papers have been published concerning the volume of endocrine glands under normal, experimental and pathological conditions. A study of the literature indicates that a number of methods have been used f o r the determination of the volume of glands. Most of the volumetric measurements however, have been made by projecting serial sections of the glands on paper and either measuring the area of the sections with a planimeter or by using the paper weight method suggested by Hammar ( ’14). Both of these methods are time consuming and expensive and even though a correction is made for shrinkage a number of other sources of error are present which may affect the final volume to a considerable degree. F o r these reasons it appears to be desirable to call attention to three inexpensive, rapid and accurate methods of volumetric determination. One of the methods to be discussed is new while the other two have apparently been neglected. METHODS Available methods for ascertaining the volume of fresh glands fall into three general categories : 1. The simple water displacement method in which the amount of water displaced by the gland may be measured by volume or bj7 weight. I \\ish to express iny sincere appreciation to Dr. R. E. Seaminoii of the University of Minnesota for constructive criticism and eouasel. Thanks are also clue the members of the department of biochemistry of this institution f o r heIpful siiggestions and for placing a Christian Beeker Chainomatic analytical balance a t m y disposal. 71 I l l E \NATO\ITCAI R > C f l R D , \OL 7 4 , NO. 1 A ND SUl’l’L. NO 1 72 C . A. SWINYABD 2. The water displacement method based on Archimedes principle in which the volume is obtained by subtracting the weight of the gland in water from its weight in air. 3. The method whereby the volume may be obtained when the weight of the gland in air and the specific gravity of the tissue are known. The simplest method of obtaining the volume of a fresli gland is to place the tissue in a known quantity of water in a graduated cylinder and determine the amount of water displac,ed. This procedure is reasonably exact for a large gland but the writer has found it very difficult to measure accurately the amount of displaced water of a small gland such as the thyroid of a rat. I n an attempt to obtain the volume of r a t thyroids with this procedure the smallest accurately graduated cylinder of suitable caliber offered great difficulty in correctly reading the change in the meniscus. When using this method, the weights when divided by the volume obtained resulted frequently in a specific gravity less than unity yet the glands would sink rapidly ~7he11placed in distilled water. However, Scammon ( '38) has improved the water displacement procedure by developing a micro-method of measuring change in the meniscus of a column of fluid which is accurate to within 0.004 cc. Another method of measuring the displaced water is by weight. This is most readily accomplished with a ppcnometer. Carlson ( ' 3 7 ) used this method to obtain the voliinic of the suprarenal glands of white mice. I n this procedure the gland is first weighed in air and then a pycnometer is filled with distilled water and weighed. Finally the gland is placed in the pycnometer which is then filled with water and weighed. The volume of the gland is then equal to the weight of the gland in air plus the weight of the pycnometer filled with distilled water minus the weight of the ppcnometer containing the gland and water. The writer has found this method to require the utmost care in order to obtain accurate results. The capillary tube of the pycnometer must be very small. All traces of moisture should be removed from the outside Y O L U M E O F E N D O C R I N E GLANDS 73 of the bottle and in addition a temperature correction must be applied. I n my experience this procedure proved t o be less accurate than the two following methods. The method based on Archimedes principle has been used snccessfullp by Stoeltzner ('06) to sliow the effect of fixation 011 the volume of an organ. If it i s undcsirahlc t o place tlie fresh tissue in water, normal saline map be used and the volume computed by means of the following formula as used hy Stoeltzner : x - (P - cl) x 100 I; Where : X = Volume of the gland p =Weight of the gland in air a =Weight of the gland i n ph~siologicalsaliiir R = Wciglit of 100 cc. of physiological saline solutioii This method also requires the greatest care hecanse a slight error in the weight of the gland in water will result in considerable error in volume. F o r example, in a 25 mg. r a t thyroid an error of only 0.5 me. in tlie weicht in water will introduce an error of 2.1176 in the volume. The water offers some resistance to the movement of the gland when weighing and for this reason it is recommended that the numerical figure used as the weight in water should he an average taken from at least five weight measurements. The weights of the glands can be obtained in a very short time if the balance is equipped with a magnetic damper. Although the density of the water in which the glands are weighed varies with the temperature, the error introduced by this variable is negligible. By assuming the density of water to be unity and makiny correction f o r the absolute density a t the highest observed temperature an error of only 0.28% was introduced in the final volume. I n the third volumetric method to be discussed the gland is carefully weighed in air, then the specific gravity determined and the volume obtained hy dividing the weight by the specific gravity. The specific gravity is determined by a modification of the method which Hammerschlag (1892) devised for the 74 C. A. SWINYARD determination of the specific gravity of blood. As far as the writer can ascertain this procedure has never been adapted for use in volume determination of glands. The method consists of placing the gland in the center of a solution of glycerol and water of such density that the gland neither sinks nor rises to the surface. When such a condition prevails it is assumed that the density of the Auid and the gland is the same and the specific gravity of the fluid is then obtained with a hydrometer or pycnometer. I n this study the specific gravity of the glycerol-water mixture was determined by means of a precision hydrometer. The accuracy of the hydrometer was checlied by determining the density of the same fluid first, by means of the hydrometer then by use of a pycnometer. The average per cent difference in the specific gravity in ten trials amounted to 0.20%. An error of this magnitude results in a volume difference of only 0.04 cu.mm. in a 25 cu.mm. rat thyroid and was not considered to be significant. Temperature variations also need not he considered with the third method. Bosart ('27) has shown that the true specific gravity of a 20% aqueous glycerol solution at 15°C. is 1.04930 while the same solution at 25°C. has a specific gravity of 1.04830. Therefore a change of 10°C. (18°F.) brings about a n error i n final volume of only 0.10%. I n view of this small difference i t is believed that the density of water can be assumed to be unity and ordinary temperature variations are, therefore, unimportant. The hydrometer xsed in this investigation was calibrated at 60°F. and the scale graded in 0.002 intervals. A uriiiometer has a suitable range for most glands and is graduated at 0.001 intervals. I n a test a urinometer was found to be satisfactory when checked with a pyciiometer and a hydrometer which was accompanied with a bureau of standards certificate. A correction factor can be applied to the urinometer readings. I n the routine use of this method a series of test tubes are filled with glycerol-water mixtures of such proportion that the difference iii specific gravity hetween adjacent tubes is 0.002. The gland is placed on a mire spoon and lowered to V O L U M E O F ENDOCRINE GLANDS 75 the center of the column of fluid. The spoon is quickly lowered to the bottom of the tube. If the gland sinks or rises to the surface it is removed, the excess fluid is blotted off and the gland then placed in a more appropriate tube. The specific gravity of the fluid in which the gland neither sinks nor rises to the surface is then measured with a pycnometer or a hydrometer of known accuracy. DISCIWSION The accuracy of the method based on Archimedes principle and the weight-specific gravity method mas tested by applying both procedures on a series of ten fiber cylinders and twenty human, dog and rat endocrine glands. The cylinders were machined to 0.002 of an inch and the true volume of each cylinder was obtained arithmetically ( nr2h) from measurements made with a micrometer. The volume of the cylinders ranged from 1579.9 cu.mm. t o 13.1 cu.mm. and mas used as a t m c volume with which the volumes obtained by the abovementioned methods were compared. The results are shown in table 1. The greatest difference between the triie volume and the observed volume was 1.63% with an average of 0.54% difference. I n seven of the ten cylinders the standard error of the greatest difference in volume was of no statistical significance. I n 70% of the cylinders the volume obtained b;v the weight-specific gravity method was closer t o the true volume than that obtained by Archimedes principle. The specific gravity of the cylinders obtained by glycerol-water suspension averages within 1% of the specific gravity obtained by dividing the weight by the arithmetic volume. I n the cylinders in which the high specific gravity required a concentrated glycerol solution or extended beyond the glycerol range the specific gravity was checked by suspension in a chloroform-ether mixture. I n 90% of the cylinders the standard deviation, probable error and coefficient of variation was smaller in the weight-specific gravity method than Q, -a TABLE 1 1 volume diff ereiice in cubic millimeters 20.3203 Maximum volume diff ereiice 0.40 C'oefficient of variation in per cent 0.018 Weight in air in milligrams 1961.3 Weight in water in milligrams 387.66 Volume in cubic millimeters froiii Archimedes principle 1573.6 Probable error in cubic millimeters 50.3853 Standard deviation in cubic millit0.5712 meters CYLINDER NUMBER 11.505 7.646 15.298 7.622 11.304 11.457 7.517 7.637 5 20.3137 k0.4222 0.091 0.271 425.1 198.3 80.64 43.2 155.77 t0.2847 7.633 2.097 1.229 1.229 k0.3674 f0.2629 0.11 0.18 0.15 0.09 20.2867 k0.1662 k0.1095 0.031 20.2898 0.731 49.7 10.2 39.60 k0.1954 7.641 2.668 8 1.263l 0.726 20.2870 3Z0.1459 0.64 k0.1081 20.0340 0.086 0.103 39.35 104.57 k 0 . 0 7 2 9 20.0229 1.275' 20.1939 0.186 104.12 39.49 k0.1307 "0.1935 20.1862 0.179 133.3 29.2 103.99 20.1255 5.090 5.100 7 k 0 . 2 1 9 1 C0.299:) 0.42 0.36 20.1568 0.100 &0.2464 0.046 310.4303 50.2826 0.040 0.036 1.273' 20.1166 0.075 155.32 t0.1037 1.235 -+0.3610 0.104 697.70 528.29 344.04 165.9 t0.2902 20.1906 f 0 . 1 6 6 1 k0.0738 1.064 k0.4317 0.081 20.4857 e0.3498 0.050 0.041 163.5 527.36 697.44 344.46 155.12 k 0 . 3 2 7 t 20.2359 -1-0.2911 k 0 2434 ?0.07 86 k0.5509 20.3921 k0.3769 0.056 0.047 0.071 837.9 241.5 649.5 160.46 78.04 121.94 698.14 165.5 344.34 528.35 k0.3715 k0.2644 20.2542 kO.2115 4 3 2 20.0313 0.236 13.21 10.0211 1.262' 50.1166 0.875 13.32 50.0786 0.468 16.6 3.3 ?O.O(ilfj 13.16 k0.0415 2.633 2.523 1-63 1.21 3Z0.0982 t 0 . 0 6 5 7 20.1058 0.403 26.20 k0.0713 1.261' k0.1356 0.520 26.06 20.0914 k0.1421 0.536 33.0 7.0 26.49 k0.0958 5.101 2.571 10 Comparison of t h e uolzime of ten macltined fiber cylindem as obta n e d b y a r z t h i n e t i c ?nemure, Arclizinedes p ~ i n c f p ' eund by specific qravzty. A11 figuies r e p r e s e n t an aceruge o f fire meusicrements 77 VOLUME OF ENDOCRINE GLANDS in the other method. These figures indicate the greater uniformity and accuracy of the weight-specific gravity method. The application of these two procedures to the endocrine glands is shown in table 2. The volume of the glands ranged from 5740.8 cu.mm. t o 18.3 cu.mm. The maximum difference in volume was 1.8% in the case of one rat thyroid gland. TABLE 2 Comparison of t h e volume of eitdacrine glands as determined b y Archimedes principle and weight-specifio gravity method GLAND WEIGHT I N AIR N GRAMS WEIGHT N XXTER N GRAMS VOLUMdl N OUBIC OENTIMETERS jPECIFIC PRAVITY Human suprarenal Hnnian suprarenal Human suprarenal Human suprarenal Dog thyroid Dog thyroid Dog thyroid Dog thyroid Dog suprarenal Dog suprarenal Dog suprarenal Dog suprarenal Rat suprareiial Rat suprareiial Rat suprarenal Rat suprarenal Rat thyroid Rat thyroid Rat thyroid Rat thyroid 4.1916 5.9237 3.8 7 57 3.8923 1.4056 0.5894 1.0307 0.9386 0.8699 0.8473 0.5521 0.8693 0.0421 0.0330 0.0348 0.0329 0.0281 0.0241 0.0230 0.0191 0.1460 0.1829 0.1597 0.1466 0.0904 0.0247 0.0600 0.0567 0.0344 0.0348 0.0205 0.0354 0.0024 0.0013 0.0011 0.0016 0.001.5 0.0013 0.0013 0.0008 4.0456 5.7408 3.7160 3.7457 1.3152 0.5647 0.9707 0.8819 0.8355 0.8125 0.5316 0.8339 0.0397 0.0317 0.0337 0.0313 0.0266 0.0228 0.0217 0.0183 1.038 1.036 1.041 1.041 1.068 1.039 1.053 1.057 1.040 1.040 1.037 1.038 1.043 1.039 1.035 1.039 1.046 1.052 1.038 1.038 VOLUME I N CUBIO CENTIMETERS 4.0381 5.7178 3.7230 3.7390 1.3161 0.5672 0.9788 0.8879 0.8364 0.8147 0.5324 0.8374 0.0403 0.0317 0.0336 0.0316 0.0268 0.0229 0.0221 0.0164 W R CENT FFERENCE N VOLUME 0.19 0.41 0.19 0.18 0.07 0.45 0.83 0.68 0.11 0.28 0.16 0.42 1.50 0.00 0.30 1.00 0.80 0.50 1.80 0.55 The average difference was 0.52%. This close agreement is believed to be indicative of the reliability of the met,hods. The weight-specific gravity method is more accurate and is easier to handle. These procedures do not interfere with subsequent histological study. Although the section method must be used when the volume of a lobe or the medulla of a gland is desired, the weight-specific gravity volume can be 78 C. A. SWINYARD quickly obt,aiiied a n d serve as a check for true volume or shrinkage correction. CONCLUSIONS 1. The commonly used projection method for volumetric determination of glands is time consuming and expensive. 2. The water displacement method is inaccurate with small glands. 3. The volume of a gland can he determined within 2% either by use of Archimedes principle o r by computing volume from weight and specific gravity. 4. The specific gravity of a gland can be determined within 1 % by suspending the gland i n a glycerol-water mixture and determining the specific gravity of the fluid with a p p i o m e t e r or a hydrometer of known accuracy. 5. The volume of a gland can be most accurately determined by the -\T.eiglit-specificgravi t p method. LITERATURE CITED EOSART, L. W., AND A. 0. SNODDY1927 New glycerol tables-tables f o r specific gravity and per eelit of glycerol-thermal expansion of aqueous soliitioiis in terms of specific gravity. J. Ind. and Eug. Chemistry, 1701. 19, pp. 506-510. CARLSON, H., E. GUSTAFSSON AND K. L. MOLLER 1937 Quantitative uiikromorphologische Studieii uber die Nebeiiiiiereii einjahriger weisser Miiuse uiiter besoiiderer Beriicksichtigung voii Geschlechtsverschiedeuheiten. Srparat ur Upsala Lakareforeiiiiigs f orhaiidlinger. Ny foljd, Ed. XLIII, S. 49-62. I I A i i h f m , J. A. 1914 hlethode, die Meiige der Rinde und des Marks der Thymus, Sonic die Anzahl mid die Grosse der Hassalsclicii Korper zahlenmiiqsig festuzustellen. Zeitschr. f. aiigcwaiidte h a t . ti. I(oiistitutioiislehre, Rd. 1, S. 312-396. HAXNEESCHLAG, A. 1692 Ein iieue Methode zur Bestimmuiig des speeifisclien Gewichts des Blutes. Zeitschr. f. Klin. Med., Ed. 20, S 444-456. SCAMMON, R.E. 1938 Personal communication. STOELTZNER, HELEN 1906 Der Einfluss der Fixierung auf das Volumeu der Orgaiie. Ztschr. f. Wissenscliaftliche Mikroskopie und f u r hlikroskopisclie Technick, Rd. XXITI, S. 14-25.