This study was supported in part by the Food and Drug Administration, Center for Veterinary Medicine, Grant No. FD-U-000059-2.

Abstract

Plasma volume (PV), extracellular fluid volume (ECFV), and total body water (TBW) were estimated in the adult channel catfish by indicator dilution techniques. These experiments were performed using catheterized, non-sedated catfish restrained in flow-through Plexiglas environmental chambers. Marker substances used were Evans blue dye for PV, sodium thiocyanate for ECFV, and antipyrine for TBW. Total body water was estimated by desiccation to a constant weight for comparison purposes.

Plasma volume was calculated to be 28.1+6.2 ml/kg body weight (mean+SD). Extracellular fluid volume was 183.2+34.6 ml/kg, and TEN was 531.3+76.4 ml/kg. Total body water -estimated by desiccation was 664.1+50.8 ml/kg.

Introduction

Accurate interpretation of the pharmacokinetics of a drug or compound in the biologic system requires knowledge of the fluid volumes of that system. These volumes represent fluid reservoirs through which drugs may disperse due to concentration, blood flow, solubility, and other factors.

Estimation of fluid volumes involving repeated blood sampling following accurate intravenous administration is difficult to manage in the unsedated fish. Infusion or sampling errors could produce marked variability in fluid volume estimations because even small inaccuracies in marker substance injection or sampling are magnified in animals with small blood volumes such as the catfish.

This investigation involved estimation of PV, ECFV, and TBW by indicator dilution using catheterized, adult catfish restrained in a flow-through plexiglas environmental chamber. Also, TBW was estimated by desiccation to a constant weight for comparison.

Materials and Methods

Thirty adult, 0.9 to 2.1 kg, catfish of either sex were randomly assigned as follows: 6 fish each to groups for determination of PV, ECFV, and TBW by indicator dilution, and 12 fish for TBW determination by desiccation.

The 18 fish used for determination of PV, ECFV, and TBW by indicator dilution were instrumented for chronic blood sampling in a similar manner. The fish were anesthetized in an aquarium with an aqueous solution of 3-aminobenzoic acid ethyl ester (Sigma Chem Co., St. Louis, MO) at a 1:20,000 dilution (w/v), removed from the aquarium, and cannulated via the dorsal aorta using polyethylene tubing (0.86 mm i.d. x 40 cm) and d 16 ga needle. Following catheter anchoring, the fish were placed in a 10 x 10 x 50 cm flow-through plexiglas environmental chamber The fish were allowed up to 4 hours after cannulation for stabilization and recovery from the anesthetic before experimentation. All studies were performed with a water temperature of 23+0.5°C.

Plasma Volume

Evans blue dye (T-1824) was injected from a standard dye solution. A separate calibration curve was prepared for each fish. Spectral absorption curves were performed for plasma and standard dye solutions. Calorimetric detection of Evans blue dye in catfish plasma was determined to be from 615-620 nm. Detection at 620 nm was utilized in the study.

Following injection of Evans blue dye, blood samples were taken at 0, 10, 20, and 30 minutes postinjection into plastic, heparinized tubes. After centrifugation, plasma samples were colorimetrically assayed at 620 nm.

Extracellular Fluid Volume

Sodium thiocyanate (SCN) was injected from a standard solution. Detection of SCN was by the method of Eder (1), and samples were assayed at 460 nm. Samples were taken at 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 hours postinjection.

Total Body Rate

Antipyrine (AP) was injected from a standard solution to estimate TBW. Following injection, fish were sampled at 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 hours postinjection. Quantities of AP in plasma samples were determined by a new HPLC assay technique developed in our laboratory (2).

Quantitation of Fluid Volumes

After individual sample values were determined, semi-logarithmic plots of plasma concentration vs. time were prepared for each fish. Regression analysis was utilized to determine the Y-intercept or zero time value. Standard pharmacokinetic calculations (3) were utilized to determine the fluid volume values. Finally, these values were normalized for body weight, and reported as mean+SD.

Total Body Water by Desiccation

For comparison to indicator dilution methods, 12 fish were assayed for TBW by desiccation to a constant weight in a drying oven (100°C).

Results

Body fluid volumes determined by indicator dilution techniques and TBW determined by desiccation are presented in Table 1. All values are reported as mean±SD.

Table 1. Body Fluid Volumes in Channel Catfish

Discussion

Measurement of fluid volumes in a variety of marine and fresh-water fish have shown differences among fish and especially differences between fish and mammals. The plasma volume in brook trout varied from 30 to 39 ml/kg depending on water temperature (4), whereas plasma volume and extracellular fluid volumes were reported as 13 ml/kq and 400 ml/kg, respectively, in catfish (5). Extracellular fluid volume and total body water have been reported as 187 ml/kg and 680 ml/kg in the channel catfish (6). Values determined in the current study closely parallel the study by Cameron (6), although plasma volume was not determined in the Cameron study.

The differences between total body water values determined by indicator dilution and by desiccation are noticeable in the current study. These differences can be explained by method and time frame variation between experiments. Indicator dilution techniques probably underestimate total body water unless the experiment is extremely protracted because circulation to deep body tissues (bone, fat) would require a lengthy equilibration period. Because the desiccation to constant weight procedure required approximately 2 days for each fish, a higher total body water percentage could probably be measured by the desiccation method.

The experimental method used to collect data in the current study has proved useful as a chronic blood sampling procedure. Other studies of interest could include determining the effect of temperature changes on body fluid volumes. Also, changes in dissolved oxygen concentration or other water quality perturbations and their effects on body fluid volumes could be explored using the described experimental method.

References

1.  Eder, H.A. Determination of thiocyonate space. M-ethods -Med. Res., 4:48-52 (1951).

2.  Kitzman, J.V., Martin, J.F., Holley, J.H., Huber, N.C. Determination of antipyrine in catfish plasma using high performance liquid chromatography. Am. J Vet. Res., submitted.

3.  Gibaldi, M., Perrier, D. Multicompartment models. In: Pharmacokinetics. Marcel Dekker, New York. 1982, pp. 45-109.

4.  Houston, A.H., DeWilde, M.A. Environmental temperature and the body fluid system of the fresh water teleost - 111. Comp. Bioche . Physiol., 28:877-85 (1969).

5.  Prosser, C.L., Weinstein, J.F. Comparison of blood volume in animals with open and with closed circulatory systems. PhyA.iol. Zool., 23:113-24 (1950).

6.  Cameron, J.N. Body fluid pools, kidney function, and acid-base regulation in the freshwater catfish Ictalurus Runctatus. J. Exp. Biol., 86:171-85 (1980).