WATER ABSORPTION AND CHANGES EN PLASMA OSMOTIC PRESSURE AS DETERMINANTS OF THE SATIATION 0F THIRST Thesis for the Degree ofTM. A. MICHIGAN STATE UNWERSTTY CHARLES THOMAS BENNETT 1946-9 UHESIS 9"..— ¢..,.( ‘H ‘- "‘ “ “RW‘Mrg. } .. , LIBR '4: R. ‘f U131 g3, ~. 3'11"}. F V alumna a, r 9 "0A6 a sane ‘ .Boux amnm mc, in gammy ”Horns . f T uxvl ‘3'." v O.ln|.ll|’lltt ABSTRACT WATER ABSORPTION AND CHANGES IN PLASMA OSMOTIC PRESSURE AS DETERMINANTS OF THE SATIATION THIRST by Charles Thomas Bennett Historically, the cessation of drinking was thought to be brought about primarily by stomach distension. It was believed that absorption of water into the blood from the gut was too slow to effect humoral changes by the time an animal stOpped drinking. It was the purpose of this thesis to measure the amount of water absorbed and the changes in plasma osmotic pressure that actually occurred by the time an animal stopped drinking. It was necessary to first establish a criterion of the satiation of drinking. In Experiment I of this thesis, 12 albino rats were placed on a 23.5 hr water deprivation schedule for 10 days. After their intake rates during the 0.5 hr access to water were monitored, the following be- havioral definition of satiety was offered: A rat was con- sidered to have stopped drinking when its intake rate was equal to, or less than, 0.2 ml/min for three minutes. In EXperiment II, water absorption from the gut into the blood and changes in plasma osmotic pressure when animals reached the satiety criterion were measured. To monitor these changes, 78 albino rats were divided into Predrink groups, rats which had no access to water on a given day of deprivation: Stopdrink groups, animals which were permitted to drink to the satiety criterion; and, Postdrink groups, animals which were permitted to drink for 0.5 hr. The amount of water absorbed into the blood from the small intestine and plasma osmolality were measured on Day 0, 1, 2, 5, and 10 of a 23.5 hr water deprivation schedule. It was found that (a) by the time rats stop drinking, approximately “.5 ml of water had been absorbed, and (b) that their elevated plasma osmotic pressure had lowered to approximately 29 libitum levels. The coincidence of the reduction in plasma osmotic pressure to ad libitum levels and the cessation of drinking support a cellular rehydration explanation of satiety. Approved: _@ 4 14m Date: ll-lf"’ Glenn I. Hatton, Chairman John I. Johnson Lawrence I. O'Kelly WATER ABSORPTION AND CHANGES IN PLASMA OSMOTIC PRESSURE AS DETERMINANTS OF THE SATIATION OF THIRST By Charles Thomas Bennett A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF ARTS Department of Psychology 1969 ACKNOWLEDGMENTS I wish to acknowledge the supportive and critical comments of Drs. G.I. Hatton, J.I. Johnson, and L.I. O'Kelly. But, especially, I would like to thank Dr. G.I. Hatton who patiently endured while I learned some of the fundamentals of water regulation. I want to also thank C.R. Almli, who as a senior graduate student, helped clarify the goals of this research. And, lastly, I wish to thank my wife, Diane, who endured these last few months better than I. 11 TABLE OF CONTENTS ACKNOWLEDGMENT . . LIST OF TABLES . . LIST OF FIGURES . . LIST OF APPENDICES . GENERAL INTRODUCTION Experiment 1 . . Method Subjects Procedure Results Discussion EIperiment 2 . . Method Subjects Procedure Results Discussion REFERENCES . . APPENDICES . . 111 Page ii iv vii O\O\U'\UxU\ U! l" Table 1. LIST OF TABLES Page Mean decreases in plasma osmo- lality in mOsm from Predrink to Stopdrink and Postdrink levels. Also represented are the percent de- creases in plasma osmotic pres- sure from Predrink levels to Stop- drink and Postdrink levels. Pre- drink, N=2hg StOpdrink, N=243 and, Postdrink, N=2n. Plus and minus one standard error is indicated. . . . . 22 Mean amount of water ingested by the Stopdrink groups (szu) as a function of days on a 23.5 hr water deprivation schedule. Plus and minus one standard error of the mean is indicated. Mean time to reach the satiety criterion for the StOpdrink groups as a function of days on a 23.5 hr water deprivation schedule. Plus and minus one standard error of the mean is indicated. Stopdrink, N=2u. . . . . 2h iv LIST OF FIGURES Figures 1. Mean amount of water drunk by 12 animals, in three minute blocks, as a function of days of adaptation to a 23.5 hr water deprivation schedule. . . . . A cumulative fibt of the mean amount of water drunk by 12 animals, in three minute blocks, as a function of days of adapta- tion to a 23.5 hr water depriva- tion schedule. . . . . . . . Cumulative amount.drunk while 12 rats adapt to a 23.5.hr water deprivation schedule as a function of the percent of total daily intake 0 O O 0 O 0 O O The Pre-, Stop-, and Postdrink mean plasma osmolality as a func- tion of the days of adaptation to a 23.5 hr water deprivation schedule. 5g Libitum (starred hexa- gon), N=6g Predrink, N=2hz Stop- drink, N=2hg and, Postdrink, N: 2“. Standard errors are indicated by the flags. . . . . . . . The mean amount of water remain- ing in the stomach and small intestine, and mean amount of water absorbed by the Stopdrink groups (N22h) and Postdrink groups (N=2b) as a function of the days on a 23.5 hr water deb- rivation schedule. Standard errors of the mean are indicated by the flagS. o c o o o o 0 Tap; The Pre-, StOp-, and Post- drink mean protein concentra- tions as a function of the days of adaptation tola-23.5 hr water deprivation schedule. Ad Libitum (stacked point), N=63 Predrink, N=243 V Page 11 18 26 Figures Page StOpdrink, N=2hg and, Postdrink, N=2h. Standar errors are indicated by the flags. Bottom: Relative plasma volumes of the Ad Libitum, Predrink, StOp— drink, and Postdrink groups as a function of days of adaptation to a 23.5 hr water deprivation sched- ule. This panel is the inverse of the top panel, indicating that as plasma protein concentration in- creases, plasma volume decreases. . . 29 Top: Mean time when rats in the plasma experiment and criterion experiment stOpped drinking as a function of adaptation to a 23.5 hr water deprivation schedule. Bottom: Mean amount drunk when rats in the plasma and criterion experiments stopped drinking as a function of adaptation to a 23.5 hr water deprivation schedule. Plasma experiment, N=2u, and cri- terion experiment, N=12. Standard errors are indicated by the flags. . 32 vi LIST OF APPENDICES Page APPENDIX A: Apparatus . . . . . . . . #6 APPENDIX B: Raw Data . . . . . . . . #8 vii GENERAL INTRODUCTION As recently as 1967, the satiation of thirst has been explained in terms of an "early" and a ”permanent” compo- nent (Adolph, 1967; Holmes, 1967).' The ”early" component, mediated by gastric and oral factors, was considered to ef- fect the actual cessation of drinking; whereas, the ”perma- nent” component, mediated by humoral factors, effected the cessation of thirst. Oral and gastric factors have been most thoroughly studied. It was assumed by Cannan (193“) that the degree of dryness, or wetness, of the mouth and throat determined whether or not an animal would drink. In 1961, Gregerson and Cizek.concluded, after reviewe ing studies comparing thirst and salivation, that a decrease in salivation was a concomitant of thirst. 'However, Bellows and van Hagenen (1939) reported.that dogs drank normal amounts of water after denervation of the mouth and throat. And, in 1965, vance reported that desalivated rats drank normal amounts of water when fed hydrated food. Apparently, then, dryness or wetness of the mouth is not a primary determi- nant of the cessation of thirst. Gastric factors (primarily stomach distention) are not believed to be a primary determiner of satiety (Adolph, 1967; Holmes, 1967). However, they have been demonstrated to at least modulate-the rate of ingestion. Montgomery and 2 Holmes (1955) inflated balloons in the stomachs of dogs, which received hypertonic saline injections. These animals did not drink for 20-40 minutes, after which time they would consume normal amounts of water, albeit over longer periods of time. On the other hand, after denervation of the stomach, by either vagotomy or total sympathectomy (Holmes and Greg- ersen, 1950; Towbin, 1955). dogs consumed normal amounts of water. Since animals could adequately regulate their fluid balance without oral or gastric cues, it was believed that humoral factors were the ultimate determiner of satiety (Adolph, 1967; Holmes, 1967; and, Towbin, 196A). There are, however, two humoral factors, a volumetric and osmometric one, which could presumably.effect.satiety. Volumetric factors might "quench" thirst as a result of an increase in blood volume resulting from the absorption of water. This increased blood volume would be detected by vascular stretch receptors which.would "signal" the animal to stop drinking. In 1968, Corbit increased bleed volume by intravenous injections of hypotonic solutions, while rats were drinking. Reportedly, these rats stopped drinking. However, other work by Corbit (1967, 1968) disputes the role volume might play. While rats were drinking, he injected serum and isotonic Ringer's solution intravenously. And, although he reportedly increased blood volume 20%, these rats continued to drink. Apparently, then, it is not merely 3 an increase in blood volume, pg; 52; that will effect the cessation of thirst. Hypothetically, osmometric factors would effect satiety as the result of absorbed water reducing the osmotic pres- sure of the surround of cells in the brain, which "signal” the satiation of thirst. There is evidence to indicate that ionic concentration of body fluids do decrease, as a function of ingested water being absorbed into the vascular system. In l93h, Baldes and Smirk reported that after man ingested water, osmotic pres- sure of the blood decreased. In 1962, Novin reported de- creases in electrical conductivity of the brain (indicating a decrease in ionic concentration), while rats were actually drinking. However, he believed that.absorption of water was too slow, and that this decrease in ion concentration in the brain resulted from ”...electrolytes moving into the gastro- intestinal tract to maintain osmotic constancy” (p. 151). In 1969, Hatton and Almli suggested that humoral explanation of satiety was possible. They also inferred from their data that it was possible that a rat actually stOpped drinking when its plasma osmolality reached approximately ad libitum levels: "...indeed, this is evident for Day 1 when rats stop drinking shortly before the 0.5 hr access period ends...,” (p. 212) and, when their plasma osmolality had actually reached an ad libitum level. It appears, then, that osmotic pressure does decrease following ingestion of water. However, as recently as 1967, )4 many believed, as did Adolph (1967), that water absorption was too slow to effect changes in body fluids by the time animals actually stOpped drinking. But, as Holmes and Mont- gomery (1960) stated, ”The mechanism by which this is ac- complished or the time interval required need to be estab- lished"(p. 911). That is, the amount of water absorbed and the level of plasma osmotic pressure, when drinking ceases, need to be measured. It was the purpose of this thesis, then, to examine more fully the osmometric component of satiety. However, it should be noted that a necessary condition for an ce- mometric explanation of satiety is that sufficient quanti- ties of water must be_absorbed in order to lower plasma os- motic pressure to ad libitum levels (or, at least some set point) by the time animals stOp drinking. Before this analysis could proceed, it was important to establish a reliable behavioral definition of satiety. This problem was the basis of Experiment I. Then, in Experiment II, changes in plasma conditions when rats reached the satiety criterion were measured. EXPERIMENT I Satiety is normally considered to have occurred when intake ceases. But, during its waking hours, an animal normally will drink for a short period, stOp, drink again, stop, and so on. Given this, the question arises: How long does an animal have to stOp drinking before satiety can reasonably be considered to have occurred? However, apparently no reported studies have attempted to behavorially define satiety. It was the objective of this study, then, to establish a criterion by which satiety could be specified in rats. Method ‘Subjects Twelve naive, male, albino rats approximately 100 days old were housed in individual cages, under conditions of constant light. They were fed Mayne Mouse Breeder Blox and given water ad libitum for three days prior to thedbegin- ning of the experimental treatments. Procedure After being adapted to their home cages, the rats were placed on a 23.5 hr water deprivation schedule. During their 0.5 hr access period to water, they were placed in drinking boxes (Described in Appendix A) without food. I Beginning on Day l.and.continuing through Day 10 of deprivation, minute by minute water intake records were taken for the first twelve minutes of the period, and then 6 every.three minutes thereafter. Immediately following the 0.5 hr access period, they were returned to their home cages. Results Figure 1 shows the mean amount of water drunk in three mdnute blocks, as they adapt to a water deprivation sched- ule. Figure 2 represents a cumulative plot of these data. Figure 3 shows the cumulative amount drunk as a function of the percent of total daily intake. After these rats drank at least 80% of their total in- take, their rate of intake dropped sharply. Prior to that point, their average rate of intake was 1.5? ml/min. In all cases, except on Day 1, this lowered intake rate was maintained for at least three consecutive minutes after 80% of a daily intake was reached. Discussion Ghent (1957) indicated that rats on a deprivation schedule tend to spend more time at a water spout in the early part of an access period. And, it is apparent from these data that as rats adapt to a 23.5 hr water deprivation; schedule that they tend to drink more and more water in the early portions of their access period. Also, from these data a behavioral definition of sa- tiety can be offered. This definition is based on the following two facts: a) coincident with a sharp drOp in the rate of ingestion, the rats drank 80% of their total daily intake: b) except for Day 1,a11.rats reduced their intake Figure 1 Mean amount of water drunk by 12 animals, in three minute blocks, as a function of days of adaptation to a 23.5 hr water deprivation schedule. 62$.on mo mueazi on .8 cu .u o. o. a. a e n 0 Wm“ M... .w «/ \. zu» :3 I . . A mi“. :3 0 03h >0.05. 21 During the adaptation to deprivation, then, Predrink plasma osmolality stabilizes at an elevated level, while Stopdrink plasma osmotic pressure remains around ad libitum levels. In contrast to this, Postdrink plasma osmolality tends to become lower over subsequent days of adaptation. Table 1 shows the decreases in plasma osmolality in mOsm from Predrink levels to Stopdrink and Postdrink levels. Also represented in this table are the percent decreases in plasma osmotic pressure from Predrink levels to StOpdrink and Postdrink levels. The mean amount of water ingested in ml/100 g of body weight is reported in Table 2, as a function of days of de- privation. One-way analyses of variance were computed on the means of the Stop— and Postdrink groups, across days of adaptation. The differences among the means of the Post- drink group were significant (F = 8.143, df = 3/20, p(0.001), as were the differences among those of the StOpdrink condi- tion (F = 0.966, df = 3/20, p<0.001). In Table 2, the mean time to reach the satiety criterion is also reported. It is interesting to note here, that al- though they had drunk different amounts when they stopped, the times at which they stepped did not differ significantly (F = 1.194, df = 3/20, p)0.025). ,In Figure 5, the amounts of water absorbed in m1/100 g of body weight.are also graphed. One-way analyses of var- iance computed cn.these groups, across days of adaptation, yielded F = 0.135, df = 3/20, p)0.25 for the Stopdrink treat- ment: and, F = 3.324, df = 3/20, p¢0.05 for the Postdrink 22 Table 1 Mean decreases in plasma osmolality in mOsm from Predrink to Stopdrink and Postdrink levels. Also represented are the percent decreases in plasma osmotic presSure from ' Predrink levels to StOpdrink and Postdrink levels. Pre- drink, N=24: StOpdrink, N=24: and, Postdrink, N=24. Plus and minus one standard error is indicated. Pre Pre Pre Pre Actual decrease of plasma osmolality in mOsm. to to to to StOpdrink Postdrink 23 Table 1 Day 1 4.2iO.8 Day 2 Day 5 8.511.8 11.3:1.6 10.511.3 18.2il.9 20-71109 Percent decrease of plasma osmolality. Stopdrink Postdrink Day 1 1,410.3 3.4:0.4 Day 2 2.610.6 5.%¢0.7 Day 5 3071005 6.710.6 Day 10 15.212.6 230Z1203 Day 10 4.910.8 706:007 24 Table 2 Mean amount of water ingested by the Stopdrink groups (N=24) and the Postdrink groups (N=24) as a function of days on a 23.5 hr water deprivation schedule. Plus and minus one standard error of the mean is indicated. Mean time to reach the satiety criterion for the StOpdrink groups as a function of days on a 23.5 hr water deprivation schedule. Plus and minus one standard error of the mean is indicated. Stopdrink, N=24. 25 Table 2 Mean amount of water ingested. StOpdrink Postdrink Day 1 12.111.0 16.2iO.6 2 16.QiO.5 17.QiO.7 5 1701i101 Zloui006 10 18.9i0.9 20.7iO.6 Mean time to reach the satiety criterion. Day 1 Day 2 Day 5 Day 10 10.311.O 10.510.6 8021005 9.610.6 26 Figure 5 The mean amount of water remaining in the stomach and small intestine, and mean amount of water absorbed by the StOpdrink groups (N=24) and Postdrink groups (N=24) as a function of the days on a 23.5 hr water deprivation schedule. Standard errors of the mean are indicated by the flags. —-_”-—__..—._ —..- _- PLASMA PROTEIN G/IOOML RELATIVE PLASMA VOL. DECREASE 27 O PREDRINK O STOPDRINK I POSTDRINK l 2 5 DAYS OF ADAPTATION IO 28 group. .Also graphed in Figure 5, is the mean amount of water remaining in the stomach and small intestine as a function of days of adaptation to deprivation. Caecal and colonic weights were measured to determine if, in fact, an appreciable amount of water enters the vascular system from these organs. It appears, however, as O'Kelly, Falk, and Flint (1958) indicated, that no ap- preciable amounts are absorbed from the caecum and colon during the time periods examined. A 3 x 4 factorial anal- ysis of variance was computed on the total fluid loss from both these organs (taken as one) of the different groups. The mean total weight losses were not significantly dif- ferent as a function of time of sampling (F = 1.025, df = 2/60, p)0.25): but, they were significantly different as a function of days of adaptation (F = 3.970, df = 3/60, A p<'0.025). Further, there was no significant change in the magnitude of the differences among the groups as a function of adaptation, as indicated by the interaction effect (P = 0.531, df = 6/60, p>0.25). Figure 6 shows the plasma protein concentration levels for the Pre-, Stop-, and Postdrink conditions, as a function of days of adaptation. A 3 x 4 factorial analysis of var- iance was computed on these treatment conditions. The means for plasma protein concentration differed significantly not only as a function of time of sampling (F 2 20.000, df = 2/60, p<0.01), but, also, as a function of adaptation of (F = 7.923, df = 3/60, p(0.01). And, the magnitude of the 29 Figure 6 Top: The Pre-, Stop-, and Postdrink mean protein concentra- tions as a function of the days of adaptation to a 23. 5 hr water deprivation schedule. Ad Libitum (stacked point), N_6: Predrink, N: 24: Stopdrink, N224: and, Postdrink, N-24. Standard errors are indicated by the flags. Bottom: Relative plasma volumes of the Ad Libitum, Pre-, Stop-, and Postdrink groups as a function of days of adaptation to a 23. 5 hr water deprivation schedule. This panel is the inverse of the top panel, indicating that as plasma protein concentration increases, plasma volume decreases. 30 M O STOPDRINK I POSTDRIN K / TIA\VII. TIOIJ. TIOII mm 6 2 mm 8 6 4 #30 20mm reds—Ohm ommmommd z. ~5th m0 mmmtmj 1:2 \ / .01 4. 2 wszmHZ. 1.4435 2. IO DAYS OF ADAPTATION 31 differences of the Pre-, Stop-, and Postdrink groups did not change significantly as a function of adaptation to deprivation, as indicated by the interaction effect (F = 0.584, df = 6/60, p<0.25). To determine whether the differences among the means for plasma protein concentration of the Pre- and StOpdrink groups differed significantly, a 2 x 4 factorial analysis of variance was computed on these conditions. The mean plasma protein concentration levels were significantly dif- ferent as a function of adaptation (F = 7.471, df = 3/40, P_mamo 0... 20:53:34 “.0 m>