Lyons, T. P., and M. L. Riedesel. L993.

GlYcerol-induced hyperhydration: its effects on fluid comPartments in the rat Life Sciences 53: L719'L787.
Life Sciences, Vol. 53, pp.1779-t787
Printed in the USA
Pergamon Press

'

GIJYCEROIJ-INDUCED HYPERHYDRATION: ITS EFFECTS ON FIJUID COMPARTMENTS IN THE RAT

T.P. Lyons and M.L. Riedesel Department of Biology, University of New Mexico
Albuquerque, New Mexico
Sufiunary
87131(Received in final form October 4,1993)

(ISF) were investigated in the rat. Ten rats were intragastrically adminisLered either 20 mI/kg of water or 5? glycerol. The TBW, ECF, and PV were determined by 3HrO, 14C-inul-in and dye diluLion, respectively at 2 h postingestion. Urine volumes and fluid retention were measured throughout the experimenL. Fluid retention was increased by 50% wiLh t.he 5Z glycerol solution when compared to water. When the fluid compartment data at. 2 h are expressed in terms of percent of body weight, the TBW and ICF of the glycerol-treated rats exceeds that of the water-treated rats, and the values for t.he ECF, PV, and ISF were slmilar after the glycerol solution and water regimens. Glycerof solutions appear to have a greater effect in expanding TBW and ICF Lhan equal volumes of
water.

Glycerol solutions are a safe, effective means to achieve a long-term state of hyperhydration. The effects of glycerol-induced hyperhydration (GIH) on the total body waLer (TBW) , extracel-l-ul-ar fluid (ECF), plasma vo]ume (PV), intracellular fluid (ICF), and interstitial- flui-d

received some attention in the fiterature (6, 7, 13, 15). The majority of these studies are concerned with cardiovascul-ar and thermoregulatory responses to different hydration states and fail- to provide adequate data on fluid balance, comparLmenLs, or distribution (6,7, L3, l-5). A common approach to hyperhydration has been the ingest.ion of large volumes of fluid. However, this technique has limitations and has not been overly successful (6, 7, 13, 15). The primary problem being that volumes of fluid in excess of euhydrat.ion tend to be rapidly excret.ed by the renal system, thus, the expansion of TBW by most oral- vol-ume loading procedures is insignificant and short in durat-ion. However, our laboratory has reported that fluid retenLion is enhanced and subsequently a state of hyperhydration is achieved if glycerol solutions are ingested in place of water or diluLe (0.18) saline (10, 11, 19). This glycerolinduced hyperhydration (GIH) resulted in a hyperosmoLic serum, but it did not effect other blood chemistries (10, 19). The GIH also proved to be a physiological advantage during exercise in the heat CPT Timothy Lyons, USARIEM, 10 Kansas St, Natick MA, 01760-5007
Copyright o 1993 Pergamon Press
0024-320s/93 $6.00

The concept of increasing total body waLer (TBW) above euhydrated levels, which wifl be defined as hyperhydration, has

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by increasing sweat rates and attenuating core temperature elevations (10). The ability to achieve a prolonged hyperhydration without disturbance to body homeostasis could prove to be beneficial in many sj-tuations of fluid imbalance. ft could have the capability to increase one's performance and tolerance to adverse conditions such as heat stress, cofd environments, hypoxia, and micrograviLy. A more direct application to the cl-inical setting would involve inducing hyperhydration prior to the initiation of procedures which may disturb fluid balance. rn addition, solutions which act as hydrating agents also have the potential to reso1ve the common medical dilemma of rehydration. The present study was conducted in order. to determine the volume expansion of each fluid compartmenL (TBW, ECF, ICF, PV, ISF) after volume loading regimens with equal volumes of waLer or 5? gIycerol. The hypothesis was that in order to maintain osmotic equilibrium the dlstribution of the glycerol would dictate the dist.ribution of the excess fluid during GIH.
Methods
Ten male adult Sprague-Dawley rats (250-350 g) were obtained from the New Mexico BioJ-ogy Dept. Animal Resource Facility. Protocols were approved by the UniversiLy of New Mexico Animal Care and Use Committee and the NIH Animal Care and Use guidelines were strictly adhered to. Protocol: The rats were surgically implanted with chronic central venous and arterial cannulae by an aseptic technique and allowed two days to recover from the surgery. Prior to the starL of the experiment the animals were fasted for 12 h and allowed ad libi-Lum water int.ake. A11 food and waLer was withheld durinq the experiment. itself, excepL for fluids required by experimental design. Animals were administered equal vol-umes (20 ml/kg) of either waLer or a 58 glycerol sol-ution (1 g/kgr body weight) by gastric intubation with a 3" feeding needfe. Five of the rats received water and the other five the 5? glycerol solution at time zero. Fol-lowing f luid ingestion the animals were immediately placed in plastic metabol-ic caqes where they remained throughout the experiment. Fluid compartments were measured 2 h post-ingestion by the methods

described

be1ow.

Fl-uid Compart.ment Measurement Prot.ocol: The method for determining fluid compartment volumes was similar to that reported by Durkot eL af. (l-987). This technique was selected because it allowed simultaneous analysis of t.he TBW, ECF, ICF, PV and ISF. The venous cannula was util-ized as a injection site and the arterial cannu1a as a blood sampling site throughout. A limited volume of fluid (1.5 m1) was both injected and removed in order to minimize disturbances

of the fluid compartments.

The TBW was measured by injecting 800 nCi of 3HrO (New England Nuclear) in 0.2 ml physiological saline via the central venous cannula 30 mi-n after fluid ingestion. An equil-ibrium time of 90 min was all-owed at which time a 0.2 ml blood sample was obtained from t.he arterial cannula, and approximately 750 nCi of 14C-inulin

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(New England Nuclear) in 0.2 ml saline and 2 mg of t.ricarbocyanine dye (Indocyanine green, Becton lickinson) in 1 ml of saline was injected. The 14C-inulin was utilized in determining ECF by Laking 0.15 ml blood samples at 2, 4, 6,30, 60 and 90 min after inject.ion in order to calculate decay characteristics. PV was measured by determining the decay characteristics of the dye with 0.2 ml bl_ood samples t.aken at 3, 5, and 7 min after injection. The TCF and ISF volumes were calculated mathematically. A detail_ed description of the calculations are provided by Durkot et al. (1989). Analvtic Methods: Blood samples were col-l-ected in heparinized tubes, centrifuged at 2500 RPM for 10 min, and refrigerated. The plasma was analyzed for 3HrO and 14c-inufin with a liquid scintillation bet.a count.er (Tri-Carb series, Model 1500, packard Instrument Co. ) utilizing a standard program for dual labeling. Tricarbocyanine dye concentration was deLermined by measuring plasma absorbance at 800 nm with a Beckman DU 64 Spectrophotometer. A gravimetric method was utilized to determine urine volume. The fl-oor of the cages were lined with absorbenL paper liners, which were weighed to the nearest 0.001 g prior to the experiment. The liners were reweighed and replaced aL 30 min intervals. The difference in weight, after accounting for fecal- material-, was recorded as urine output. The urine output was accumulated and util-ized in conjunction with the fluid intake to calculate overall fluid balance at each hour. Stat,istical Analvsis: The data is presented as means + sLandard errors. One-way ANOVA was utifized to determine t.he effect of the fluid regimens on fluid compartment volumes. Differences in urine volume and fluid retention were anal-yzed by two-way Anova with a repeated measures model. A standard Tukey's post hoc Lest was applied to further characterize these differences. A p<0.05 resul-ted in rejection of the null hypot.hesis.

the

t.wo groups (Table 1)

Results The mean weights and ingesLed fluid volumes were similar
.

among

TABLE I ffuid compartment volumes. WATER (20 ml/ks) 5? GLYCEROL (20 m]/kg) n=5 n=5 Weight, g 362.3 + 8.1 356.8 + 6.2 7'7+O? Fluid Volume, m1 7.t + 0.2 TBW, ml 245.L + 4.5 248.3 + 6.3 ECF, ml 63.3 + 2.0 60.6 + 2-B PV, m1 16.4 + 0.2 15.6 + 0.5 ICF, ml 181.8 + 2.8 LBt .7 + 3.7 ISF. ml 45-B t 1 .8 44.9 + )..a Fluid comparLment analysis at 2-h post-ingestion. Data presented as means t SE.

Body weiqht and

urine volume and mean volume of fluid retained are presented in Figures 1 and 2. The 5? glycerol sofution resulted in reductions in accumulaLed urine outpuL at 1, 2 and 3 h when compared to ingestion of 20 ml/kg water (p<0.01) (Fig. 1). A 508 decrease was
Mean accumul-ated

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The attenuated urine output observed after 2 h (nig. 1). contributed to an increased fluid retention with the glycerol solution over that of water (Fig. 2). Differences j-n retention were 1.9 mI at t h, 2 mI aL 2 h, and 2.4 ml at 3 h (p<0.01)(Fig. 2). The effects of ingestion of water (20 ml/kg) and a 58 glycerol sol-ution (1 g/kg) on the fluid spaces are presented in Tables 1 and 2. The absolute volumes of fluid compartments at 2 h were not different between the two experimental groups (Table 1-). If these

I wnrnn flsz cLYcERoL

bD v4

Er

D
tr-

o
t'l

z t

rrME (h)

FIG
Accumul-ated

]-

urine vofume. Inqestion of 20 m1/kg of water or 5? glycerol solution (L g/kg) at time zero. Significance between waLer and glycerol at t h, 2 h, and 3 h, p<0.01 daLa are cafcul-ated as a percent of the t.otal body weight, there are increases j-n TBW and ICF with the ingestion of glycerol when compared to the same volume of water (p<0.05) (Table 2). The TBW was 67.8 + 0.3 I and 69.7 + 0.4 ? of the total body weight foll-owing ingestion of water and 5% glycerol, respectively (p<0.01-) (Tab1e 2). The ICF was 50.3 + 0.5 3 with water and 52.8 + 0.7 I with glycerol (p<0.05) (Table 2). The vafues for the other fluid compartmenLs, ECF, PV, and fSF, were similar at 2 h for the two groups (Table 2)'

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f-l
!

wnran

sz

cLYcERoL

a f-1

z
t'l
t4

F3

,t o

rrME (h)

FIG.
Volume

of fluid retained. Same fluid ingestion regimen as Figure 1. Significance beLween water and glycerol at t h, 2 h and 3 h, p<0.01.
TABLE

TI

Fluid

comparLment volumes
WATER

(? of bodv weiqht).
5Z GLYCEROL (20 ml/kg)
n=5

(20 ml/ks)
-_trJ LT_

TBW

0.1 PVZ 0.1 ICF ? 0.5 ISF ? 0.2 Data presented as means
ECF
Z

.4 t 4.5 r 50.3 r L2.9 !

11

67.8 r 0.3

69.1 t 0.4** 17.0 r 0.3 4.4 ! 0.L 52.8 + 0.7* 12.6 ! O-3 + SE, * p50.05, ** p50.01.

Discussion

Oral administ.ration of a 5? glycerol solution enhanced fluid retention and attenuated urine output for 3 h in the rat. when compared to the ingestion of water afone. This positive fluid

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balance at 2 h post-ingestion resulted in a condition of increased TBW, which to i considerable extent resulLs from a ICF expansion (Figrs. 1 and 2, Table 2). No differences in ECF, PV, or ISF were observed between Lhe two fluid loading condiLions at 2 h (Table 2). The changes in fluid balance attributed Lo Lhe GIH are in close agreement wiCh data previously observed in boLh humans and ra|s ,lnder similar protocols (10, 1'1', 19 ) ' Riedesel et al . (19 ) and Lyons et al. lro) reported urine volume reductions in humans of 40 Lo 52 ? after the ingestion of glycerol sol-utions when compared to The 50% decrease in the present an equivalent vol-ume of water. study is within the range of 45 to 53? observed in a recent inveitigation with the rat subject to identical hyperhydration regimeni (11-). The increased fluid retention also support.s the fact that a glycerol*induced hyperhydration was achieved (Fiq. 2) - If the volumes of fluid retained are calculated as a percenL of total fluid intake comparisons with our human data are more meaningful. These values from the current study are B3?, 722, and 648 at t h, 2 h, and 3 h following the 5? glycerol solution and 55?, 442, and 30? after ingestion of water at time zero. These data are similar to the 83?, JOeo, and 63? at 1, 2, and 3 h with glycerol and 51?' 412, and 25? without glycerol observed in previous work with GIH in rats (11). Humans have demonstrated retentions of B2z at 1.5 h, 74% aL 2.5 h and 68? at 3.5 h with glycerot and 403 at 1.5 h, 382 aL 2'5 h, and 35? at 3.5 h with water (10). The maintenance of a highly positive fluid bal-ance for 3 h supports the fact. that a GIH similar Lo that observed in previous rat and human investigations was achieved in the current series of experimenLs. The reported data are sugqestive of the fact that the TBW and the ICF comparLment undergo a greater expansion following ingestion of a 5? glycerol solution than with an equal volume of water. This study by aLsign only a1lows characterization of the differences in fluib -distributiorr between the two hyperhydrating regimens; therefore, absolute changes in fluid compartment volume from baseline to post-ingestion for a given fluid treatment are not known. It iJ plausible that both of the fluid protocols increase the ECF volume, but the GIH also results in an additional expansion of the ICF compartmenL, which accounts for the greater TBW. Tn this case it would be expected that with GIH the volume changes for the However, when compared to water TBW and ICF would be sj-milar. ingestion the GIH resul-ted in increases of 3.2 and 5.9 ml in TBW and tCF, respectively. This discrepancy between the volume of expansion of t.he TBW and ICF will be further discussed. The increased fluid retention with the GIH should be comparable in magnitude to the volume expansion observed in the TBW. The diffefence in fluid retention between water and the 5? glycerol was 2.0 ml aL 2 \; however, the difference in TBW between the two fluid A previous study with Lhe rat estimated that regimens was 3.2 m] th; cIH should result in a theoretical fluid retention of 3.7 mI, which would be distributed inLo the TBW (11).
The two previously mentioned inconsistencies in the data are not readily eiplained. The methods employed are well accepted and the results are comparable to other techniques (9, 16, L'1,23). The possibility of experimental error in some of the procedures exists, and in addition, the vol-umes involved in GIH in an animal- model,

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such as the rat, are relatively small. Therefore, interpretation of Lhe actual fluid compartment volume data should be ]imited, and the relative direction of change may be more important. A11 the animals in this study demonstrated a similar response to GIH, primarily.an increase in tew and rCF (Table 2). The acLual volume of expansion may be questionable for the reasons mentioned, but the trend was consj-stent and statistically significant.

Both the osmoLic properLies and distribution of the glycerol appear to play a major rote in the GIH. The hyperosmolality due to the elevat-ed serum glycerol levefs is present up to 2.5 h in humans, but the enhanced flui-d retention is maintained for 4 to 8.5 h (10, 19). Thj-s difference demonstrates that the GIH in previous studies is not merely an aftermath of a hyperosmotic vascul-ar compartment. As a result, it is unlikely Lhat Lhe GIH j-s so1ely dependent on a hormonal response, such as an elevated ADH release.

A plausible hypothesis for GIH is based on the fact that glycerol fras been sho-wn to distribute quite readily throughout the ier{ fZZ, 24). In order to maintain isoLonicity, fluid shifUs would Tlt. accompany ihe movement of glycerot into the fl-uid spaces. . fluid being distributed intraCellularly with GIH woul-d minimize the stimulation of volume and osmotic receptors, and it would Seem that a large compartment, such as the ICF, has a much greater potenLial to act as a fluid reservoir. The increase in TBW by ICF expansion would also support the idea that the fluid load is being distributed out of the vascufar space. our ]aboratory has not detected changes in PV at 1 to 8 h post-inqestion of glycerol (1 g/kg') plus a large volume of water -or the rat (10, t1', 1-9 ) . The absence of a '(21-.4 *1 Zt g) in man volume loading has al-so been observed in oLher hemodilution after studies (15). Nadel et al. (15) reported that. a study in humans involving exog.enous ADH and 2,000 ml of water resulted in an increased fluid retention and hyperhydration, but PV was not changed, which might signify an ext.ravascular fluid distribution
(15 )
.

fn conLrast to our findingis Murray et al. (:-4) reported that the ingestion of concentrated glycerol solutions during exercise did not reiult in hyperhydration, but did induce a short-term expansion of the pV. Simillr findings were observed with pre-exercise glycerol feedings (5). In these studies the increases in PV were itiributed to ai el-evat.ed plasma osmolality, which was a result of the high serum glycerol concentrations. However, it is difficul-t to relate the resulls of these studies with those of our laboratory because the protocols did not involve fLuid loading or a GIH. - The elevated plasma osmolality after glycerol infusion has also been shown to elicit a hemodilution response (1, 8, 12). A GIH similar to that observed in studies with humans was achieved in this experiment with the rat. In addition, it seems that glycerol solulions may be more effective than water in expana-in-g TBW because of the added ability to expand intracellular spaces. More extensive studies involving glycerol distribution and tire corresponding fluid shifts need to be conducted in order to further characterize the mechanisms of GIH.

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Acknowledqements
Thi-s study was taken in part from a dissertation submitted by T.P. Lyons in partial fulfillment for a PhD degree in 1991 and was

supported by

NASA Research

Grant NAG-9-401-Basic.

References

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