COLUMBIA LIBRARIES OFFSITE HEALTH SCIENCES STANDARD i i 1 lllllilillllliMMIIilllii HX64088324 QP913.B4Se1 Biochemical studies RECAP tj Columbia (HnitJer^itp mtI)fCttpofIlfttjgork CoUese of l^iiv^icims anb ^urgeonjf Hibrarp Digitized by the Internet Archive in 2010 with funding from Columbia University Libraries http://www.archive.org/details/biochemicalstudiOOseam Biochemical Studies of Beryllium Sulfate. DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIRE- MENTS FOR THE DEGREE OF DOCTOR OF PHIL- OSOPHY IN THE FACULTY OF PURE SCIENCE OF COLUMBIA UNIVERSITY BY EMILY CROMWELL SEAMAN, B.S., M.A. NEW YORK CITY. Easton, Pa.: EscHENBACH Printing Co. 1912 Biochemical Studies of Beryllium Sulfate. DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIRE- MENTS FOR THE DEGREE OF DOCTOR OF PHIL- OSOPHY IN THE FACULTY OF PURE SCIENCE OF COLUMBIA UNIVERSITY BY EMILY CROMWELL SEAMAN, B.S., M.A. NEW YORK CITY. Easton, Pa.: EscHENBACH Printing Co. 1912 ACKNOWLEDGMENT. This research has been done at the suggestion and under the personal direction of Professor William J. Gies. During its progress he has given the investigation much personal attention. I wish to express my sincere appreciation and gratitude for his interest and assistance and especially I wish to thank him for the generosity which made it possible for me to work in his laboratory. Emily C. Seaman. Laboratory of Biological Chemistry of Columbia University, College of Physicians and Surgeons, New York City. May 16. 1912. CONTENTS. Page I. Introduction 7 II. General Influences on Nutrition 7 III. Pharmacological Effects 28 a. Administration per os :^t, b. Subcutaneous injection (as beryllium sulfate) :^^ c. Subcutaneous injection (as beryllium lactate) 35 d. Intravenous injection 36 IV. Effects on Enzyme Action 41 a. Sucrase 41 b. Ptyalin 42 c. Pepsin 43 d. Trypsin 44 V. Precipitation of Protein 45 a. Egg Albumin 45 b. Proteoses 45 c. Gelatin 45 d. Acid Meta-protein 45 VI. Effect on Growth of Seedlings 46 VII. Effect on Bacterial Growth 49 VIII. Summary of Conclusions 50 IX. Biographical 52 X. Bibliography 53 feces. The raw meat was preser\,^ed in a frozen condition.^ The cracker meal was thoroughly mixed and kept entirely dry in large glass-stoppered bottles. The best quality of lard was purchased in small amounts and kept in a re- frigerator. Infusorial earth Avas used instead of bone-ash in some of the experiments in order to prevent possible con- version of administered beryllium into the insoluble com- pounds which bone-ash might induce. Distilled water was used. The dog was given all the daily portion of food at one time, the solid ingredients being thoroughly mixed with water. Periods and Weights. — Each day of the experiment ended at 2.30 p.:m. The dogs were weighed just before being fed. A new period of record was always begun with the day on which the dog was placed under new conditions. Collection of Excreta. — The lu-ine was collected each day just before feeding, and the daily volume recorded. The cage was washed ever}* second day, the washings being added to the total urine for the two days and the volume made up to 2000 cc. with distilled water. The total nitrogen was determined from this. The fecal matter was removed from the cage as soon as possible after its elimination, placed in an evaporating dish and dried as speedily as possible by exposure to the air. The feces for each day were kept separate and weighed when thoroughly dry. It was notice- able that, with the exception of a condition which will be men- tioned later in the experiment, the appearance of the material was uniformly dry and chalky, which agrees with the pre- vious experiences in the use of infusorialareth in nutritional experiments directed here by Prof. Gies. Analysis. — The total ingested nitrogen, sulfur, phosphorus and inorganic matter were determined from the food. The nitrogen of the urine was determined on the total volume for every two days and the nitrogen in the feces for each period. The ash, sulfur and phosphorus of the urine and feces were de- ^ Gies: American Journal of Physiology, 1901, v, p. 235; Gies and collaborators: Biochemical Researches, 1903, p. 69 (Reprint No. i). termined for the period. The urine was preserved with thymol. The feces were finely powdered before analysis. Analytical Methods. — The total nitrogen was deter- mined by the Kjeldahl process, oxidation being effected with concentrated sulphuric acid and a little cupric sulfate. The total sulfur and phosphorus of the urine were determined by the caustic alkali fusion methods. Because of the pres- ence of silicate in the feces it was necessary to remove silica after fusion before determining the sulfur and phosphorus. This was done by dehydration with acid followed by desic- cation, extraction and filtration. The total inorganic mate- rial was obtained by evaporating the sample to dryness and charring at a low redness until all the carbonaceous material had been burned and only a white residue remained. Beryllium Sulfate. — The beryllium sulfate used in the metabolism experiments was a ptu-e product obtained from Dr. Charles L. Parsons for this special work. First Metabolism Experiment. — The dog selected for this experiment was a healthy female bull terrier weighing about twelve kilos. Diet. — The diet was the same throughout the experiment except that bone-ash was used during the fore and first dosage periods. This was then changed and infusorial earth used in its place for the remainder of the metabolism work, for the reason stated above (Table I) . lO ^ ^ On HH 00 g O 00 'tl- -i rh ,•; 00 w ■« -fi 00 2 O ^ S ^ O ^ S « C5 M (N 00 00 O O 00 cs O O w M HH "ij i^ W s ^ ,x 8 ►J "d

. oo NO O O w •3 o S a « Si, ^ Si, •« s Preparatory Period. — Bight days were sufficient for the preparatory period. During this time the animal became accustomed to the food and environment. The weight became nearly constant. The preparatory period ended on Nov. 24, 19 10 at 2.30 o'clock and on that day the experi- mental work was begun. It was continued uninterruptedly until 2.30 o'clock on December 29, 19 10 — (36 days). The experiment was divided into six periods of different lengths and conditions as follows: Fore Period. — Normal conditions. Days, 1-8. Nov. 24 to Dec. I, 1 9 10. During this period entirely normal condi- tions were maintained. First Dosage Period. — Influence of beryllium sulfate administered per os. Days, 9-16. Dec. 2-9, 1910. On the ninth day the first dose of beryllium sulfate was given. The dosage was started with o.i gram and increased each day until on the last day of this period 5.0 grams had been administered. The dose was always given just before feeding by enclosing the beryllium sulfate in a ball made of part of the weighed meat. The mass was swallowed quickly without mastication and the remainder of the food given at once. No symptoms were produced by this amount of beryllium sulfate but it was decided not to prolong this dosage period. There was a slight decrease in weight. Special Period. — Days, 17-18. Dec. lo-ii, 1910. Be- cause of a continued loss in weight and variations in the volume of urine it was decided to consider the two days following the dosage period as a special period. Intermediate Period. — Days, 19-24. Dec. 12-17, 1910. During this period there was a tendency toward restoration of normal nutrition. The weight still decreased but toward the end of the period it began to increase and the volume of urine struck a normal average. Second Dosage Period. — Days, 25-28. Dec. 18-21, 1 9 10. After an interval of eight days beryllium sulfate was again administered. As in the first dosage period the amount was started with o.i gram. It was increased each day until by the fourth day, 3.6 grains of beryllium sulfate had been given. At this point marked effects were produced. Previous to the time this amount was given (2 p.m., Dec. 21) there were no signs of sickness. At 9 a.m. the next morning (Dec. 22) a large amount of vomit consisting of undigested meat and mucus was found on the pan and in the urine. Some of the material on the pan was dry indicating that there had been an attack of vomiting sometime between midnight when the dog was last inspected and 9 a.m. There were no other signs of sickness. The dog's nose was cold and she seemed full of life. The urine containing the vomit was strained and the solid particles from this and from the pan were weighed and added to the next day's food (Dec. 22). In anticipation of vomiting the urine receiver had purposely been kept free from thymol. The volume of urine was de- cidedly increased, but this was probably due to the presence of the vomit. Just before the time for feeding on the same day the dog vomited twice. This vomit consisted of about 25 cc. of colorless froth with a little liquid. It was allowed to remain on the pan to be added to the urine. No more beryllium sulfate was given. The maximum amount given in this experiment was evidently a toxic dose for this dog and could not be satisfactorily exceeded in such work. The beryllium sulfate had probably exerted a deleterious effect on digestion and was possibly diuretic. The feces for this period were black and soft in marked contrast to those of the former periods. After Period. — Days, 29-36. Dec. 22-29, 19 10. Although no more beryllium sulfate was given, the dog showed a tendency to vomit on the first day of this period (Dec. 23). At II A.M. and again at 12 m. she ejected small quantities of thin liquid. There were no solid particles in the vomit at either time. The material was added to the day's urine. The dog was very lively, the nose was cold and she showed no other signs of sickness. During this after period there was a gradual but constant increase in weight. Analytical Results. — The data in the daily records and 13 the summary of results with the daily averages for the first metabolism experiment are given in Tables II-IV. Discussion of Results. — During the fore period the animal showed a fluctuation in weight ending in a slight gain. The period was marked by a decided plus balance of the nitrogen, ash and phosphorus and a small plus balance of the sulfur. It was evident that the diet was more than suffi- cient for the dog's requirements. The first dosage period was without marked effect although a total of over 5 grams of beryllium sulfate were given. The weight fluctuated slightly during the period but there was very little loss at the end. There was decrease of nitrogen and sulfur elimi- nation but an increase in elimination of ash. The phos- phorus remained practically the same. There was an in- crease in the volume of urine. It seemed quite evident from the data for the two periods following (and later experiments bear this out) that the beryllium sulfate was cumulative in its influence. During this time there was a decided loss of weight and further increase in the volume of urine. The first two days were so marked in these respects that the excreta were analyzed for a separate period. The results showed a great loss in nitrogen, ash, sulfur and phosphorus suggesting that the beryllium sulfate had produced decided nutritive distiu-bance. During the following six days (the intermediate period) there was some return to normal conditions as was shown by the decreased elimination of nitrogen, sulfur and phosphorus. In the second dosage period nearly 4 grams of beryllium sulfate were given in four days. It had marked toxic effect, producing vomiting. Up to this time the feces had been hard, chalky, and uniform in character, but on the third day of the period, they began to be dark and decidedly soft. This condition lasted until the second day of the after period. There was a marked loss of weight and an increased elimi- nation of nitrogen and sulfur during this second dosage period but as in the former dosage and after periods, the effect of the beryllium sulfate extended beyond the time of 14 the administration. Here there was a still greater loss of body nitrogen, sulfur and phosphorus. The result of the first experiment showed a decided loss Date. Nov. 24 25 26 27 28 29 30 Dec. I Body weight. Kilos. 11.82 11.82 11.82 11.80 11.84 11.84 11.84 11.83 Tabl,e; II. Experiment I. Fore Period. Urine. BeSO* Gram Vol. cc. 437 431 445 435 405 510 435 Reaction, Sp. gr. litmus. 1017 acid 1015 acid 1016 am. ac* 1015 acid 1015 acid 1015 acid 1014 acid 1015 acid Nitrogen. Grams. 12.07 12. Il 11.55 12.53 Feces. N Dry weight. Grams. 15.4 31.7 26.6 20.2 14.2 40. 5 none 37.0 First Beryllium Sulfate Period. 2 3 11.83 1 1 . 76 0.1165 0. 2131 470 483 1015 1014 am. ac. am. ac. II 22 5-5 36.0 4 5 11.78 11.84 0.2131 0.2581 470 475 1015 1015 acid acid 12 87 lost 17.3 6 7 11.78 11.80 05034 I. 0012 433 506 1014 1016 acid acid 10 03 20.2 16.5 8 9 11.79 11.80 I . 2473 I .5002 483 418 1016 1017 acid acid 12 42 17.0 350 10 II 11.79 II .69 special Period. 480 1017 acid 550 1015 am. ac. 16.16 ^3.5 14.5 * Urines that were amphoteric but apparently more acid than alkalin are referred to as "am. ac." in the Tables. 15 Table H — (Continued). Intermediate Period. Date. Nov. 12 13 14 15 16 17 Body weight. Kilos. Urine. 71 68 69 69 73 70 BeSOi. Grams. Vol. cc. 423 425 475 470 445 Sp. gr. IOI6 IOI6 IOI5 IOI7 IOI5 IOI5 Reaction, litmus. acid acid acid am. ac. acid acid Nitrogen. Grams. 13 59 13 03 12.03 Feces. Dry wei^t. Grams. 25 -5 14.0 20.0 II 5 14 5 17.0 Second Beryllium Sulfate Period. 18 II 75 1003 464 1015 am. ac. 19 II 74 5039 425 1016 am. ac. 20 II 77 I Olio 473 1018 am. ac. 21 II 61 2 0100 595 1016 am. ac. 13 32 14.27 10.5 235 II 5 18.5 22 23 24 25 26 27 28 29 63 68 73 74 77 77 80 82 [fter Period. 415 446 1020 1017 am. am. ac. ac. 15 00 21 5 II 5 433 490 1017 1016 am. am. ac. ac. 14 21 22.0 14.0 455 455 1019 1019 am. am. ac. ac. 14 83 19 5 20.0 415 468 1016 1017 am. am. ac. ac. 14 27 8.0 13.0 i6 as d ^ vo ^ V 2 ■a So •i ^ . t-H 1— ( o 1 .Sm l-H e o W cq i •4 P3 1 1 J< >^ ^ ^ c^ -i^ ^ o ■g . 1 ^ a- •i!» V w " %> 1)00 <^ pq H +-• •2 3 X) o o °? 0^ ON ON 1 I M O CO ON 00 W '4- O M 00 On O M CM ON CS o d + + 00 00 00 o 00 01 00 r^ 0\ o o \0 r^ 00 CN CO Tf On lO O + T 00 00 On On rOO VO M O M 00 \o O O rlr iTi + + IT) tJ- NO ro On On lO m Si o p o c c O O u u o On M ^ + + CO O COVO 00 tJ- a> ^8 2 $5 "^ G fl > lU c3 C O O >^ fa >-* 1-1 ra »0 cs 00 CN| O CO CO ON w CO M VO CO Tt- M lO rr O M 00 ■^ Tt- cs + + "^ o Os >0 r}- -^ CO O) MM M O M M M _J.^ O 'ii- M 00 On CO ^^ ^';^Q CM ON CO 00 u^ CO M 00 C^ + + 00 M On CO ■^ CO M r^ 2223 J en en t« c/i 01 17 Tj- >-l O ex ^^ I I 41 ■* n •d O ( a) a V oi ^s? •o-o a^ a l 00 vO 00 o CM ro O « 0) Td- -^ CO ID t-i VO vO 00 rl- 1-1 00 00 00 « 6 « 6 O 6 M 9 9 + + 11 o 00 M 6 ^ d VO 00 (S CS "^ ID 1-1 O CD 1-1 in 0\ w " r? ?? 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PW h-l M M C» 00 00 M 00 ■* 0) 3 O O\00 £^ • ■ • ■3 1; « '^ o 00 cS 4) a (N O ri- £*t! cs • • ■ a H^ hH 1-1 M '^QO to r^ 10 10 lO :5" '-' o 00 00 00 rO CD CO 10 o 00 11 00 O I-. o 6 6 6 ■rhoo 00 cs M 11 000 00 vo 00 o d 00 ■^ O 00 ^O -^ ro 000 >0 vo ro ONOO rj- fO '^ '^ 000 CS M 10 CS CS Cv« >^ O O O cs cs cs 00 10 t^ \o t^ t^ O 00 vo O vo 10 00 r^ t^ '^ ^ ^ o u ,2 >. CO >, ^'^^ -a 00^ 00 ^00 .2^-2 O 4> CI 23 Discussion of Results. — The fore period showed that the animal continued to gain weight on the prescribed diet as in the former experiment. During the period in which 5 grams of beryllium sulfate were administered there was a fluctuation in weight which continued into the after period and a slight decrease in the total volume of urine. The daily volume varied considerably but these variations usually at- tended pronounced sickness as the result of the dosage. The dosage period showed a marked increase in the elimina- tion of nitrogen and phosphorus, but the sulfur and total in- organic matter were not much affected. The after period showed a continued and increased elimi- nation of body nitrogen. The weight of the animal fluctuated during this time but by the end of the period there was a re- turn to normal. There was an increased elimination of sulfur but less phosphorus than in the dosage period. The data for the analysis showed the same nutritive disturbances as in the first experiment although not to so marked a degree. Third Metabolism Experiment. — The animal used in this experiment was a medium sized short haired dog weighing nearly lo kilos. Diet. — The diet was of the same character as in the two former experiments. Table IX. Composition of Daily Diet. Experiment III. Lean Cracker Infusorial Ingredients. meat. meal. Lard. earth. Water. Total. Days I-18 I-18 I-18 I-18 I-18 Grams. Gram. Grams. Grams. cc. Grams. Daily Amount 160.0 0.40 30.0 7.0 350 587 Nitrogen 6.4 0.88 0.0018 7-3 Sulfur 0.30 0.06 0.36 Phosphorus 0. 16 0.05 0.21 Inorganic mat- ter 1.6 0.32 6.79 8.71 24 Preparatory Period. — This period consisted of 15 days. It was two or three days before the dog became accustomed to its new environment and ate all the daily amount of food. During this time there was great fluctuation in the amount of daily urine. On the last day of the period there was com- plete retention of the urine and the large volume on the first day of the fore period was the extra elimination. Fore Period. — Normal condition. Days, 1-6. May 16- 21, 191 1. With the exception of the fluctuation in the daily volume of urine normal conditions were maintained through- out the period. Dosage Period. — Days, 7-12. May 22-27, 1911. On the 7th day beryllium sulfate was given per os just before feeding as in the previous experiments. The dosage was started with 0.5 gram and gradually increased until on the third day of the period the dog received i gram. This was followed by very slight vomiting. The same dose was re- peated on the following day. Slight vomiting resulted the next day at noon. This was frothy and unformed. On each of the two days following the dog received i gram of the sulfate with the food. The vomit as a result each time was stringy and mucous-like. The decided increase in amount showed that the dog was at the point of very marked effect from the beryllium sulfate and the dosage was stopped. After Period. — Days, 12-18. May 28 to June 2, 191 1. During this period normal conditions were maintained. Analytical Results. — The analytic data pertaining to this experiment are recorded in Tables X-XII. 25 Date. May. i6 17 18 19 20 21 Body weight. KUos. 9.60 9.68 9.70 9-73 9-75 9- 63 BeSOi. Gram. Table X. Experiment III. Fore Period. Urine. Vol. cc. 475 250 320 355 355 455 Reaction, Sp. gr. litmus. IOI9 IOI5 1020 IOI7 IO17 1020 acid acid acid acid acid acid Nitrogen. Grams 13 05 12.86 14.2 Feces. Dry weight. Grams. 7.0 none 12.2 8.4 10.6 19.2 Beryllium Sulfate Period. 22 9 23 9- 24 9 25 9- 26 9- 27 9 .65 0.5049 310 1020 acid .66 0.7543 300 1 02 1 acid .72 1.0055 340 1017 acid .65 1.0293 440 1019 acid .65 0.9890 400 1020 acid .75 1. 0000 275 loio acid 12.6 143 154 9 II 9 II 8 13 After Period. 28 9.65 29 9.68 30 9.64 31 9.69 June 1 9.86 2 9.68 525 102 1 acid 325 1015 acid 390 1020 acid 325 1020 acid 220 1 05 1 acid 490 1020 acid 15.0 14 3 143 19 7 9 8 10 15 I noi 20 le 2 26 to rr l-H 00 vO vo o « M3 fO r^ M lO ID 00 r^ w c< CTJ O O »o rO O vo o lO (N . ^^ O lO r^ On ■<*• CO c» O M HH O o cn w O vo NO Tt- vO O '^ o rOO 00 N NO NO ■^ M r-". lO NO w CO CO -H-H cs On w d CM d CD 9 d T3 O '^ •* >D "^ + + 1 1 to' 6"^ mo ^' M 00 CO r-o e V CO ID '^ ro C) w CO 1 CS HH ^ u d On CD O '^ w rr> l-I « 1 o "3 i 1 .4 1 s rc ^ NO ON t^ On ^ NO NO »o t:: ■•X) Sf2 r 00 On ON en "+> cs 00 to CO ^ HH '^ CO O £h i-^ 2 CO M M 6 o n CM 00 HH o^ CM CM 9 d s i •n oi ^ rl- + + IM in ^ + + 1 1 art ^ 1 4, -a "« S^ M Oi On NO o ■^ fa M 00 NO CN i-i CO ,H (^ d W CM d NO NO -^ CM O d d + + On *-• 00 w O O oq O NO -« CM ■<^ • O CO CO O O «DO0 CM O M O ^ 3 ^ food urine feces ^Cn 3 S m foe m uri m fee a a a GOO be -g 3 S 0. o a; •-< CJ 4) U3 2 P P ^ '*-"^ So »:; v; v; «■♦-( t+-( <+H 2 ^3^ =3 a a a a aJ en w M c3 3 =3 ?5 V^ trogen trogen trogen > h from h from h from lance lily ave If ur fro Ifur fro Ifur fro Ifur fro 01 o 3 J3 0) > osphori osphori osphori lance ,ily ave •r-< •r-l -r-l CO Oi t« CO eft cc «J 3 3 3 3 mmw.m 13 ca Jl J3 J3 C8 OT 27 r 35 1o2 00 O »^ 'O lO^ O rO ro O o o o o o o o o o ON 00 C4 O »0 rD CO -^ g 00 !Z lO On X S I. 1^ ^ §: S ^ ^ja -^ -^00 q'S r^ tj- Tj- ^5 vo cpr^ I _. , 2 lO On O rt g c; (s 00 CM o^ 2 ■ • ■ H 0,-S W O M ■g J, \0 CO irj £f g 6 6 6 6(1 "-I f5 vO o . • . *J O c< CO •^ Tj- Tl- ^ 6 O >o IT) -; CM O CM O CM C^ CM o o o M M C^ O O O 6 6 6 00 -^ CO CO »0 lO uovO t^ CO C^ ^ o o o vo c >i ?! t«x!r-i d 1/2 CO +j t/3 -■ o o "5 '^ C v-( ^-> +j C O X o O 0) o i O O _0 J>-i a rt O S a 03 4) 03 C 0) (^^a 03 rt tfl X o 4) (U ■ p:; t=^ J3H 03 J, c t3 0) c n c 03 1; i-' « lU to o o3 J:; s w ts i I 3 OS bo ^ C 2 c n ^ 03 42 -H O o3 "^^ bS ^ 03 a o J3 o lU fti S M bi) iS J bO I 31 ^ ^ S S o 3 3 b C « §.§«-« 03 •-^'O -M O '-' .3 ■<-' +-< C X > iH cS 3 t— ' en o C • HH ^ ^ ■M 6 "^ a di crt 5i tn JS ctf C