Acta Physiol Scand 1982, 116: 235-238

51Cr EDTA determinations of the extracellular fluid volume in hemorrhage: a study with fed and starved rats

JAMES WARE, KARL-AXEL NORBERG, MIKAEL NORMAN and GUNNAR NYLANDER Department of Surgery, Karolinska Hospital, Stockholm, Sweden

WARE, J., NORBERG, K.-A., NORMAN, M. & NYLANDER, G.: "Cr EDTA determi- nations of the extracellular fluid volume in hemorrhage: a study with fed and starved rats. Acta Physiol Scand 1982, 116: 235-238. Received 9 March 1982. Department of Surgery, Karolinska Hospital, Stockholm, Sweden. Extracellular fluid volumes have been determined in fed and 2630 h starved rats, before and after 60 min of hemorrhagic hypotension at 70 mmHg. Bilateral renal vascular ligation was carried out to prevent clearance of the isotope tracer used, "Cr EDTA. The time taken for a bolus of the tracer to distribute itself in its space, was determined in fed and starved animals after the standard period of hypotension. This was found to be 37 min for fed animals, and 50 min for those which had been starved. When the isotope ECF space was compared before and after hemorrhage in fed animals, it was unchanged, despite an estimated blood volume loss of 39%. The isotope ECF space was reduced 5 . 5 % after hemorrhage in animals which had been starved. It is concluded that fed rats mobilised intracellular fluid to the interstitium in hemorrhage, whereas, starved animals did not. This finding of variable fluid homeostasis is ascribed to the different nutritional status of the animals investigated, and has considerable implications.

It has been predicted that an intracellular fluid (ICF) shift to the interstitium should occur to ac- complish full restitution of the blood volume (BV), during or after hemorrhage (Pirkle & Gann 1975). An osmotic effect mediated by a state of hypergly- cemic hyperosmolality has been proposed as one of the mechanisms which could cause such a fluid shift (Ware et al. 1980).

Non-starved rats subjected to hemorrhagic hypo- tension develop a state of hyperglycemic hyperos- molality, which is not observed in rats starved 24-30 h (Ware et al. 1982). The occurrence of an ICF shift to the interstitium in fed animals, and its absence in those which had been starved, was in- vestigated after a standardised hemorrhage. 5'Cr EDTA has been considered a useful isotope tracer for measurements of the extracellular fluid (ECF) volume (Virgilio et al. 1 9 7 0 ~ ; Larsson 1980). The choice of experimental method is limited by the use of the rat. Double equilibrium methods are lengthy (Virgilio et al. 1970a, b) . Therefore, a double injec- tion method was used (Roth et al. 1969). To shorten the time of multiple sampling after hemorrhage, the

distribution times of the isotope tracer were first determined in fed and starved animals. These data were incorporated into the sampling protocols of animals used to determine the ECF space before and after hemorrhage.

METHODS Male Sprague-Dawley rats (n=35), were divided into 4 experimental groups (A-E). Fed animals, groups A and C had access to a standard pellet diet and tap water ad libitum up until the time of the experiments. Animals in groups B and D, were placed in metablolic cages 24-30 h before experiments, with only access to water. All rats were weighed immediately before use.

A commercially available neuroleptanalgesia was used, fentanyl (0.315 mg x ml-') and fluasonium (10 mg x ml-'), given im., 0.5 ml x kg-' b.wt., being reinforced after 30 minutes with 0.1 mlxkg-' b.wt. S.C. A tracheos- tomy was performed on all animals, which breathed room air. Both renal vascular pedicles were ligated via a small laparotomy. PE 10 and 50 catheters were established in the right jugular vein and femoral artery respectively. N o heating pad, or temperature control is required when us- ing neuroleptanalgesia in rats (Ware et al. 1982). When experimental preparation was completed, heparin, 500 IU x kg-' b.wt. i.a. was given.

Arta Physiol Srond 116

236 J . Ware et al.

T +*..

I =3.88-0~0012x

I I I 1 0 30 60 90 120

M I N U T E S

Fig. 1. Percentage residual plasma activity of an injected bolus of "Cr EDTA given to fed "anephric" rats after 60 min of hemorrhagic hypotension at 70 mmHg, group A. Broken line = distribution curve, solid line = clearance curve, intercept = equilibrium. All values are means k SE, n=6.

Groups A (n=6) and B (n=6) were used to determine the equilibration time of a tracer bolus given after 60 min of hemorrhagic hypotension at 70 mmHg. Groups C (n=12) and D ( n = l l ) served to determine the isotope spaces before and after the standard period of hemorrha- gic hypotension.

All animals were subjected to a standard period of hypotension. The short arterial catheter was connected to a three way tap system open to a pressure transducer (Siemens-Elema, 746) and recorder (Mingograf, 803, Sie- mens-Elema), for continuous recording of the mean arteri- al blood pressure (BP). A heparinised syringe was con- nected to the third coupling and used to withdraw blood until a constant BP of 70 mmHg was obtained, which usually took 5-10 min. No autotransfusion was allowed. The final hemorrhage volume was noted, and the hemato- orit of the withdrawn blood obtained. Using this hemato- cnt, corrected for plasma trapping with a factor of 0.98, and a water content factor for plasma of 0.929 (Altman & Dittmer 1%1), the plasma water loss of the hemorrhage was calculated.

Isotope equilibration and space determinations Groups A and B received a 0.1 ml bolus "Cr EDTA solution i.v., and duplicate sampling was performed there- after every 15 min for 2 h (Larsson 1980). Blood sampling was strictly standardised and duplicate hematocrits ob- tained at every occasion.

To determine the ECF spaces before and after hemor- rhage, a 0.05 ml bolus was given prior to hemorrhage and 0.15 ml bolus after. This measure was taken to reduce the significance of remaining background activity for the sec- ond space determination. All samples and injections were weighed. 4 duplicate samples were taken at 5 min inter- vals after 40 min equilibration, for the initial space deter-

Acm Ph?siol Scund 116

mination (Larsson 1980). After obtaining background samples, 4 duplicate samples were taken at 10 min inter- vals after equilibration of the second tracer bolus (40 rnin for fed animals, and 50 min for starved animals).

Multiple standards prepared in the same volumes as the samples were always counted together with the samples in a double channel gamma well scintillation counter (Selek- tronik, model, 54-22). In groups A and B, posthemorrhag- ic isotope equilibration times were determined from plas- ma distribution and clearance curves. These were ob- tained from the quotients of injectid activity and averaged sample residual plasma activites. Plasma clearance of -"Cr EDTA in rats, with ligated renal vascular pedicles, is considered to fit a linear function (Larsson 1980). Thus, the intercept of the best fit of residual activities and those preceding has been considered to be the time of equilibri- um. The isotope space determinations, before and after hemorrhage, for groups C and D, have been obtained from zero extrapolations of lines of least squares for the residu- al plasma activities, according to a linear function. Back- ground activities were subtracted from post hemorrhagic determinations, and the plasma trapping and plasma water content factors, were used to obtain the istope distribu- tion volumes. Results are given as means k SE.

RESULTS

Groups A and B BP for group A was 95k4.1 mmHg and group B 922 1.6 mmHg. Their hemorrhage volumes were similar, A, 1.5620.15 ml x 100 g-' b.wt. and B, 1.79t0.24 mlxI00 g-l b.wt. After the period of hemorrhagic hypotension and during the time of repeated sampling the BP fell 5 f2 .4 mmHg in both groups, during this two hour period. Fig. 1 and 2 show the residual plasma activities expressed as a percentage of the injected dosex100 g-' b.wt. The intercept of the broken (distribution) and solid lines (clearance) represents the best estimate of equilibri- um. This was 37 min in group A (fed) and 50 min for group B (starved).

Groups C and D The initial BP was similar, group C (fed), 95k4.3 mmHg and D (starved), 96k5.1 mmHg. The BP changes noted during the posthemorrhagic period were the same as for the animals in groups A and B. Table 1 shows the fluid balance data for both groups. The hemorrhage volume in group C was 39% of the estimated blood volume (blood volume =6% b.wt). The calculated plasma water loss being 7.3 % of the prehemorrhage isotope (ECF) volume. The final isotope ECF volume after the hemor- rhage, however, was unchanged. Group D animals, which had been starved for 24-30 h, sustained an estimated BV loss of 37%, or a plasma water loss

Extracellular fluid volume in hemorrhage 237

Y . 4 0 4 -0.001JX

W

I I I I 1 0 30 60 90 120

M I N U T E S Fig. 2. Percentage residual plasma activity of an injected bolus of ”Cr EDTA given to starved “anephric” rats after 60 min of hemorrhagic hypotension at 70 mmHg, group B. Broken line = distribution curve, solid line = clearance curve, intercept = equilibrium. A11 values are means k SE, n=6.

equal to 6.4 % of their prehemorrhage isotope ECF volume. This fluid loss was confirmed by a reduc- tion of the isotope space by 5.5% (p<0.005). The hematocrit reductions in fed animals were 43 % greater than those obtained with starved animals (pCO.05).

DISCUSSION

The isotope tracer substance, ”Cr EDTA, has been judged suitable to measure a fluid space con- sidered to represent the ECF volume (Virgilio et al. 1970a, Volf et al. 1971, Larsson 1980). All authors, however, have realised that isotope dilution meth- ods probably never delineate an anatomically dis- crete volume, and one group has introduced the concept of a functional ECF volume to avoid un- necessary confusion (Shires et al. 1973). Notwith- standing this, isotope dilution methods, using equi- librium curves obtained before and after a standard hemorrhage, have been reported to be sensitive enough to detect ECF isotope volume alterations as small as 5 % in the nephrectomised baboon (Virgilio et al. 1970a). Similar accuracy might also be pre- dicted using inbred “anephric” rats of similar size and maturity (Larsson 1980).

The starved animals in group D have decreased their ECF volume, after hemorrhage, by an amount equal to the plasma water loss, confirming several previous reports (Serkes & Lang 1966, Shizgal et

al. 1967, Roth et al. 1969, Crystal & Baue 1969, Virgilio et al. 1970b).

Fed animals after an almost 30% reduction of their BP, lost 39% of their estimated BV. This is in agreement with a larger series using the same an- aesthetic (Ware et al. 1982). Despite this hemor- rhage, which removed 7.3 % of the initial ECF vol- ume, the final ECF volume was unchanged.

A greater reduction of the hematocrit was also observed, compared to starved animals, and this is considered to reflect a larger volume of fluid avail- able to the circulation through plasma refill. This seems uneqiuvocal evidence of an ICF shift to the interstitium, and although discussed previously (Jarhult 1973, Ware et al. 1980, Gann et al. 1981), has not been demonstrated earlier.

During the initial stages of hemorrhage a state of pseudodiabetes has been described (Hiebert et al. 1973). This is characterised by a refractory or ab- sent pancreatic beta cell response to the developing state of hyperglycaemia (Halmagyi et al. 1966, Moss et al. 1970, Coran et al. 1972, Fritz et al. 1976, Carey et al. 1976), and a resistance to insulin’s effect on cell membrane transport of glucose (Chaudry et al. 1973, 1975). The net effect, is the building of a glucose osmotic gradient at the cell membrane, which in it’s turn mobilises ICF to the interstitium.

The time taken to equilibrate a bolus of 5’Cr EDTA in normotensive “anephric” rats, has been reported to occur within 30 min (Larsson 1980). After the period of hypotension, fed rats (group A) took 37 min and starved animals (Group B), 50 min.

Table 1. Fluid balance data for fed and starved animals, groups C and D, before and after hemor- rhage, mean L- SE, volumes in ml x 100 g-’ b.wt.

Group C Group D Fed Starved (n=12) (n= 11)

Initial ECF volume Final ECF volume Difference Hemorrhage volume Plasma water loss % of initial ECF

Initial hematocrit Final hematocrit Difference

volume

21.02k0.30 21 37k0.27 21.44k0.32 20.67k0.25 +2% (NS) -5 .5% (P c0.005)

2.36k0.1 I 2.20f0.13 1.53k0.08 1.39k0.09

7.3 % 6.4%

42.4k0.8 43.2k0.7 31.7k0.9 35.7k0.7 25 % 17%

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238 J . Ware et al.

These findings are corroborated by cardiovascular studies in fed and starved rats subjected to similar hypotension. The cardiac output in fed animals, late in hemorrhage, was almost 50% greater than that in starved animals. Furthermore, muscle and skin blood flow in the former animals was almost twice the level in starved animals (Quiros & Ware 1982).

In conclusion, the "Cr EDTA spaces, consid- ered to represent the ECF volumes in "anephric" rats, have been determined before and after a standard period of hemorrhagic hypotension, in fed and starved animals. Whereas, an ICF shift to the interstitium was confirmed in the fed animals, who also benefitted from a greater degree of plasma refill, no such fluid shift occurred in those who had been starved. The mechanism of this fluid shift is considered to be due to a glucose osmotic gradient building at the cell membrane, secondary to a state of hyperglycemic hyperosmolality and insulin resistance. These osmotic conditions are absent in 24-30 h starved rats. and hemorrhage causes a re- duction of their ECF space by an amount equiv- alent to the plasma water loss of the hemorrhage.

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