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Reference values of peritoneal fluid from healthy foals
#.
•
~
kJ Refereed
REFERENCEVALUESOF PERITONEALFLUIDFROMHEALTHYFOALS Eddy Behrens, MV; Maria E. Parraga, MV, MS; Alexander Nassiff, MV and Nobel DelMar, MV
SUMMARY
Thirty-two clinically healthy Thoroughbred foals, aged 14 to 75 days ~--45.5 days), were used to evaluate several parameters in peritoneal fluid. The evaluation revealed clear, pale yellow fluid with a mean total protein concentration of 1.8 gm/dl + 0.7 ~.SD). The mean total of nucleated cell count was 1,418/ul + 1,077, which was distributed as follows: neutrophils 15.3% + 20.4, large mononuclear cells 43.8% + 24.8, small mononuclear cells 22.3% + 24.5. Eosinophils were not observed in any sample and the erythrocyte count was 12,070/ul + 15,219. Nine different biochemical parameters were determined in paired samples of peritoneal fluid (PF) and serum. Results of the biochemical analysis of PF were : glucose 136.9 mg/dl + 21, aspartate aminotransferase (AST) 50 IU/ L + 18.9, alkaline phosphatase 43.6 IU/L + 15.15, lactate dehydrogenase (LDI-/)46. 5 IU/L + 22.9, creatinine 1.39 mg/ dl + 0.22, sodium 133.6 mEq/L + 2.18, chloride 98.7 mEq/ L + 4.16, potassium 4.2 mEq/L + 0.36 and fibrinogen <200400 mg/dl. When differences of these parameters for serum and PF were analyzed by paired comparisons, all were significant (P<0.05) except those for creatinine and sodium. A valuable aid for determining the probable cause of abdominal pain is analysis of peritoneal fluid (PF). Analysis of peritoneal fluid is helpful in assessing other abdominal conditions such as chronic diarrhea, parasitic larvals Author'saddress: Dr. Behrens, Large Animal Clinic, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843-4475. Acknowledgements:The authors thank Ing Rafael Branger for his financial support and Haras Las Acacias, El Bosque and Monumental for permitting us to work with their foals.
348
migrans, abdominal abscessation, granulomatous enteritis and metastatic gastric squamous cell carcinoma. This ancillary aid is even more important for foals because rectal palpation is impossible due to their small size. Abdominocentesis of the horse was described more than 30 years agoz2 and is now a routine procedure for evaluation of horses with abdominal distress. Evaluation of PF from horses with acute abdominal conditions is well documented;4,s,s,9,2°,24,a62s.~.41 however, most of the literature deals with adult horses. Although the field of equine neonatology has been recently advancing, 3° we were not able to find studies of analysis of PF from healthy foals. The serum and PF concemrations of aspartate transaminase (AST), l° alkaline phosphatase (ALP),13,16,~and lactate dehydrogenase (LDH),4° as well as the concentrations of lactic acid, 25 fibronectin, TMand phosphatea have been reported. The aim of those determinations was to search for an accurate index to predict early compromise of bowel; however, the sensitivity and specificity have been variable. ~ The purposes of this study were: 1) to report reference values for PF from healthy foals, 2) to report values of different biochemical parameters evaluated in paired samples of serum and PF and 3) to determine the relationship of those parameters between serum and PF.
MATERIALS AND METHODS
A total of 45 clinically healthy Thoroughbred foals, ranging from 14 to 75 days old ~---44.5 days old) from 3 different farms, were used in this study. The foals had not
EQUINE VETERINARYSCIENCE
Table
1.
Methods used for determination of each biochemical parameter. Test Aspartate aminotransferase (AST). Lactate dehydrcgenase (LDH) Alkaline phospharase (ALP) Glucose Creatinine Chloride thiocyanate
Conct. 0-450UV 0-1000UV 0-1200 0-400 0-10 0-130
Method Measurement of NADH/NAD Measurement of NADH/NAD P-nitrophenyl Phosphate Colorimetric glucose oxidase Colorimetric picric acid Colorimetric mercuric
Sodiumand potassiumconcentrations were measured by flame emission photometry.
been dewormed and had onlyreceived tetanus prophylaxis. The PF and blood samples were taken early in the morning before the foals were turned out to the pasture with their mares. The abdominocentesis was performed with the foal in standing position. The foals were manually restrained with 1 or 2 assistants, no chemical restraint was used. The most dependent portion of the abdomen between the exyphoid process and the umbilicus was clipped and prepared for an aseptic procedure. The operator, wearing sterile surgical gloves, introduced an 18 gauge x 2.5-cm needle into the linea alba and then slowly advanced it until fluid was obtained. If no fluid was obtained, the needle was rotated, repositioned, or aspiration with a syringe was performed. If no fluid was obtained, one additional needle was inlroduced a few centimeters from the first needle. Samples were assessed for color and coagulability. Peritoneal fluid was collected in a tube that contained ethylene diaminetetra-acetic acid (EDTA) for cytological analysis and in a tube that contained no anticoagulant for biochemical analysis. The latter sample was immediately centrifuged at 1,500 rpm for 5 minutes, and the supernatant was removed and frozen at -30°C until further analyzed. The cells in the former sample were counted with a hemocytometer. The diluting fluid for the red blood cells was Gower's solution and that for the white cell count was 2% acetic acid. Both counts were performed using a 1:20 dilution. After the cells were counted, the EDTA-containing sample was centrifuged at 1,500 rpm for 5 minutes and a smear was made from the sediment. The smear was stained with Wright's stain and a differential cell count was performed (200 cells counted). Three groups of cells were considered: neutrophils, large mononuclear cells (macrophages and/or mesothelial cells) and small mononuclear cells (lymphocytes). The estimation of total proteins in the effusion was determined by refractometry and the concentration of fibrinogen was determined by heat-precipitation at 56°C for 3 minutes. The biochemical determinations of the supernatant of PF, as well as those of serum were performed using a Boehringer Mannheim photometer. =The methods used for the determination of each biochemical parameter are listed in Table 1. =Model4010, Mannheim,W. Germany.
Volume 10, Number 5, 1990
The statistical analysis of all values except those for fibrinogen was by student's t-test for paired comparisons, b Values for fibrinogen were ordinal data and were, therefore, analyzed nonparametrically for paired comparisons. =The values were significantly different from one another at P<0.05.
RESULTS
This research initially included 45 foals, but the abdominocentesis was completed with 32 foals. Of the remaining 13 foals, PF was not obtained from seven foals, the amount of PF was insufficient from 5 foals to be analyzed, and in one foal the values obtained were indicative of an underlying disease not detected during physical examination. Therefore, the investigation was performed with 32 foals (16 females and 16 males). The cytologic evaluation is shown in Table 2. The biochemical parameters concurrently evamated in peritoneal fluid and serum were analyzed individually, the results are shown in Table 3. Values of parameters in the two samples (PF and serum) were compared and statistically analyzed with a student's t-test for paired comparison. The results are shown in Table 4. The values for fibrinogen were compared and statistically analyzed by a nonparametric method.=
DISCUSSION
Peritoneal fluid was not obtained from seven foals, which may occur in normal adult horses,a In five foals the quantity of PF was very small, in this respect the foal may have a lower rate of PF production than the adult horse in which 60 mg/hr has been reported. 27Other possible causes remain speculative but include the following: altered gravitational flow of PF (compartmentalization) by abdominal oSnedecor,GW and Cochran WG: The comparisonsof 2 samples,IN: Stalis~cal Methods, 7th edition,the Iowa State University Press,Ames, IA, 83-106,1982. cRosner B: WilcoxonSign rank test, In: Fundamentals of BiostatiMics, PWS Publishers,Boston, MA, 270-276,1984.
349
Table foals.
2
Cytologic evaluation of peritoneal fluid from 32
Peritoneal fluid Red Blood Cells (/ul) Nucleated cells (/ul) Neutrophils (%) Lg. Mononuclear cells (%) Sm.Mononuclear cells (%) Eosinophils (%) Total protein (gm/dl)
Mean 12,070 1,418 15.3 43.8 22.3
SD 15,219 1,077 20.4 24.0 24.5
1.8
0.7
viscera, misalignment of the needle with a pool of PF, restricted flow of PF by adhesions, production of PF reduced by dehydration, and inadvertent insertion of needle into the falciform ligament. In any event, the volume per se' is not a reliable indicator for assessment of PF since many times a small volume of normal fluid may be obtained in horses with compromised bowel (inguinal or diaphragmatic hernias or flcocecal intussusception). These conditions are not commonly observed in foals. Conversely, a foal with a small perforated ulcer in the stomach or the duodenum, may not show abnormal changes in PF because of omental walling-off activity.42 The intestine was not punctured during the abdominocentesis procedure although a sterile disposable 18 gnage x 2.5-cm needle was used. It has been recommended that a teat cannula be used in foals because of the increased risk of unintentional enterocentesis with a needle.3g This requires local anesthesia and a small stab incision. However, complications seldom resulted from enterocentesis, 4,2s,as if the intestinal segment that was penetrated was not distended or devitalized. TM All the samples of the PF were clear, serous and strawcolored. This physical parameter can be a rough index of the number of leukocytes present, depending on the degree of turbidity3 s The results of the cytology of the PF showed nucleated cell counts (~=1,418/ul) lower than the mean values reported for the adult horse, 4,~,2sin which up to 10,000/ul is considered normal. We found only one report describing
cytology of PF in normal foals younger than one week, but no other data was provided for comparison." A total WBC count greater than 5,000/ul was reported in five neonatal foals with abdominal distress which subsequently underwent exploratory celiotomy? We suggest that a WBC count in the PF up to 3,000/ul be considered a normal value. The presence of RBCs ~ = 12,070/ul) could have been contamination of the PF during collection, intraperitoneal hemorrhage or increased capillary permeability with diapedesis of RBCs into the PF. r The RBCs in our samples were most likely secondary to collection-induced hemorrhage, despite the gross normal appearance grossly. Eosinophils were not observed in any sample, which was unexpected because none of the foals that underwent investigation had been dewormed and some degree of eosinophilia in the PF was anticipated. Eosinophilia in PF has been supportive of a tentative diagnosis of strongylosis.4 The prevalence of Parascaris supp. infestation during the foal's first months of life and the parasite's life cycle, could influence the observation mentioned. However, the lick of eosinophils in the PF does not rule out strongylosis. 41 The lymphocytes in PF were present in low numbers (~=22.3 %) similar to adult horse values,4,~,24The predominant cells in the PF (x-+=43.8%) were large mononuclear cells (marcophages and mesothelial cells). In the adult horse lower values have been reported. 4.~,2s,38Neutrophils (R=15.3%) were less predominant than in normal adult horses.4,9,24,28
The values for protein in the PF (~=1.8 gm/dl) were similar to those of the adult horse.4,Q.2sA marked variation of protein in PF was reported depending on the method used (refractometry or chemical). A lower concentration of protein was found by refractive index, r To obtain a more accurate approximation, determinations of protein were performed by refractometry with the supernatant after centrifuging the PF for 3 minutes. The peritoneal membrane is permeable to water and most solutes, allowing a dynamic equilibrium through
Table 3. Biochemical parameters concurrently evaluated in peritoneal fluid and serum. Parameter
Peritoneal Fluid ± SD (range)
Glucose (mg/dl) AST (lUlL) ALP (lUlL) LDH (IU/L) Creatinine (mg/dl) Sodium (mEq/L) Chlodde (m Eq/L) Potassium (mEq/L) Fibrinogen (mg/dl)
136.9 +21 (100-178) 5O + 18.9 (28-120) 43.6 + 15.15 (13-82) 46.5 + 22 (23-108) 1.39 + 0.22 (0.91-1.87) 133.6+2.18 (128-137) 98.7 + 4.16 (92-106) 4.2 +0.36 (3.6-4.9) < (200-400)
Serum Y ± SD (range)
89.40 + 17.80 (59-152) 185.S0 • 45.8O (8e-433) 334.70 ±102.0 (195-667) 326.50 +75.80 (179-464) 1.39 + 0.24 (0.85-1.85) 134.3+3.38 (126-141) 96.5 + 3.63 (88-103) 4.6 +0.47 (4.0-5.6) (20O-800)
x = mean, SD = standarddeviation,AST = aspartateaminotransferase ALP = alkalinephosphatase,LDH = lactatedehydrogenase
350
EQUINE VETERINARY SCIENCE
Table
4.
Values of parameters of PF and serum samples
analyzed with a student's t-test for paired comparison. Parameter Glucose (mg/dl) AST (IU/L) ALP (IU/L) LDH (lU/L) Creatinine (mg/dl) Sodium (mEqlL) Chloride (mEq/L) Potassium (rnEq/L) Fibrinogen (mg/dl)d
Mean differencec + SD 47.50 + 30.14 -135.78 ¢ 43.03 -291.15 + 97.54 -279.90 ¢ 73.51 0.00687 ¢ 0.2634 -0.71875 ¢ 2.842 2.187¢ 3.49 -0.44437 :t: 0.4420 (range < 200-400)
P<0.05 t
=Meandifference = value in Peritoneal Fluid -value in Serum, SD = Standard deviation, * = significant, - = non significant. K)rginal data analyzed nonparametricallyfor paired comparisons (Wilcoxons Sign Rank test).
passive diffusion between blood and peritoneal fluid?3 Therefore, the PF is a dialysate of plasma and reflects any changes in the visceral and parietal peritoneum. 6,~On that basis, the concentration of diffusible elements in plasma should be very similar to those observed in PF. We measured, in paired samples of serum and PF, 9 different biochemical parameters. 1. Glucose: the mean concentration in serum was 89.4 mg/dl + 17.8 and was very similar to the ones reported for adult horses.5,9,28 Higher values have been reported for yotmgerfoals (<1 week). ~1.2~The samples were taken from our foals early in the morning, a few hours after they had nibbled the mare's feed. Blood glucose concentrations in our foals could have been affected by feeding time and alterations of light and darkness, 9 in addition to the stress related to the restraint and abdominocentesis. The mean concentration in PF was 136.9 mg/dl + 21 and was similar to values reported for adult horses,s in which the values of glucose in PF were also greater than the corresponding values in blood, s,28 2. Aspartate aminotransferase (AST): the mean concentration in serum was 185.5 IU/L + 45.8 and the concentration in PF was 50 IU/L + 18.9. These values are similar to the ones reported for adult horses in one study,28but differ from values reported in another paper. 5 We could not explain the difference. 3. Lactic dehydrogenase (LDH): the mean concenlration in serum was 326.5 IU/L + 75.8 and the concentration in PF was 46.5 IU/L + 22.9. Our values for serum are similar to the ones reported for adult horses,5,~but our values for PF were lower. 4. Alkaline phosphatase (ALP): the mean concentration in serum was 334.7 IU/L + 102 and the mean concentration in PF was 43.6 FU/L + 15.15. These values are similar to the ones reported for adult horses, s,~6but higher when compared with another paper. 28 The concentrations of these enzymes in PF were poorly correlated with those in the serum. A similar observation has been reported for the adult horse. 5 Although the enzyrnes investigated are not tissue-specific, the liver and
Volume 10, Number 5, 1990
intestines contain a considerable amount. On that basis it is feasible that gross deterioration or pathological lesions of intestine or liver may be necessary in order to observe an increase in the PF. It has been shown that the intestinal mucosal enzymes in the horse have characteristic patterns of development from early life to maturity.32Since production of the intestinal enzymes depends on the differentiation and maturation of the enterocytes, we hypothesize that our foals did not have fully mature enterocytes. Even more likely, the numbers of enterocytes were probably less than those in the adult horse. This assumption could be supported by recent evidence which confirms that intestinal length will keep increasing up to 1 year of age. ar A slow rate of cellular proliferation and migration of the intestinal mucosa has been reported in suckling rats and rabbits. ~ This could also apply to our suckling foals; therefore, the slower rate of turnover of the intestinal epithelium could partly explain the low enzymatic concentration in PF. It is noteworthy that in adult horses, ALP in PF was predominantlygranulocytic in origin; therefore, it was not a reliable indicator of small intestinal disease in horses. 16The same observation could be applied to the foal. Finally, the elevated concentration of enzymes in serum could be explained by the relatively high hepatic mass (as percent of body weight) and the high enzymatic activity per gram of hepatic tissue in young animals. 13,~8 5. Creatinine: the mean concentration in serum was 1.39 mg/dl + 0.24 and the mean concentration in PF was 1.39 mg/dl + 0.22. The concentration in serum was similar to the one reported for adult horses, but the concentration in PF was lower than that of adult horses. 28 Uroperitoneum in the foal due to tears in the bladder, urachus 2 or ureters, ~2and without concomitant gastrointestinal disease, will show an unremarkable analysis of PF with the exception of an increased concentration of creatinine. It has been shown that creatinine in PF is a reliable test for uroperitoneum in the horse27 In the foal a ratio of creatinine in PF to that in serum that is greater than 2:1 is highly indicative of urperitoneum)~ Our foals' values supported the mentioned statement since they showed a ratio of 1:1. 6. Sodium, potassium and chloride: the mean concentration of sodium in serum was 134.3 mEq/L + 3.38 and the concentration in PF was 133.6 mEq/L + 2.18. The mean concentration of potassium in serum was 4.6 mEq/L + 0.47 and the mean concentration in PF was 4.2 mEq/L + 0.36. The mean concentration ofchloridein serum was 96.5 mEq/ L + 3.63 and the mean concentration in PF was 98.7 mEq/ L + 4.16. These values were similar to the ones reported for adult horses 2°,28and foals serum. ~ 7. Fibrinogen: the range of concentrations in serum was 200-800 mg/dl. This range was slightly higher than that reported for adult horses.34 The range of concentrations in PF was <200-400 mg/dl. This value is similar to the ones reported (~=200 mg/dl) for adult horses with effusions of
351
PF without mesenteric thromboses. 14It is noteworthy that for 65% of the group subjected to this investigation, the concentrations of fibrinogen in the PF were less than 200 mg/ dl. Therefore, the majority of the foals had concentrations of fibrinogen in the PF that were either to minute to be detected or were absent. Since fibrinogen plays an important role in any inflammatory process through its conversion to fibrin, and its increased concentrations will decrease to normal as inflammation subsides, it might be a reliable test in abdominal disturbances. To conclude, when the biochemical parameters of paired peritoneal fluid and serum samples were compared and statistically analyzed, the enzymatic activity (AST, IDH, ALP) the concentration of glucose, potassium, chloride and fibrinogen were significantly different. Concentrations of creatinine and sodium were not significantly different.
REFERENCES 1. Adams SB, Fessler JF, Rebar AH: Cytologic interpretation of peritoneal fluid in the evaluation of equine abdominal crises. Comell Vet 70:232-246,1980. 2. Adams R, Koterba AM, Brown MP et al: Exploratory celiotomy for gastrointestinal disease in neonatal foals. Equine Vet J 20:54-58,1988. 3. Arden WA, Stick JA: Serum and peritoneal fluid phosphate concentrations as predictors of major intestinal injury associated with equine colic. JAm VetMedAssoc 193:927-931,1988. 4. Bach LG, Ricketts SW: Paracentesis as an aid to the diagnosis of abdominal disease in the horse. Equine VetJ 6:1161221,1974. 5. Brownlow MA, Hutchins DR, Johnston KG: Reference values for equine peritoneal fluid. Equine VetJ 13:127-130,1981. 6. Brownlow MA, Hutchins DR, Johnston KG: Abdominal paracentesis in the horse - a pathophysiological approach to interpretation. Aust Vet Pract 11:143-154,1981. 7. Brownlow MA, Hutchins DR, Johnson KG: Abdominal paracentesis in the horse - basic concepts. Aust Vet Pract 11:6067,1981. 8. Coffman JR, Garner HE: Acute abdominal disease of the horse JAm Vet Med Assoc 161:1195-1198,1972. 9. Coffman JR: Equine clinical chemistry and pathophysiology, Kansas, VetMedPublCo 201-208,1981. 10. Davis JV, Gerring FL, Goodbun R et al: Experimental ischemia of the ileum and concentration of the intestinal isoenzyme of alkaline phosphatase in plasma and peritoneal fluid. Equine Vet J 16:215-217,1984. 11. Deem Morris D: Bacterial infections of the newborn foal. Part 1. Clinical presentation, laboratory, findings and pathogenesis. Compend Contin Educ Pract Vet 6:$332-$339,1984. 12. Divers TJ, Byars TD, Spinto M: Correction of bilateral ureteral defects in a foal. J Am Vet MedAssoc 192:384-386,1988. 13. Dumas MB, Spano JS: Characterization of equine alkaline phosphatase isoenzymes based on their electrophoretic mobility by polyacrylamide gel disc electrophoresis. Am J Vet IRes 41:2076-2081,1980. 14. Feldman BF, Ziemer E, Waddle J: Effusion fibronectin concentration detect presence of mesenteric thromboses: a preliminary prospective report: In: Proc 2nd Equine Colic Res Symp, Vol 2, Athens, University of Georgia, 57-59,1986. 15. Fitts CT, Davis JH: Complications of parecentesis. In: Management of surgical complications, ed by Arts CP and Hardy JD, Philadelphia, WB Saunders Co, 227-228,1975. 16. Froscher BG, Nagode LA: Origin and importance of increased alkaline phosphatase in peritoneal fluid of horses with colic.
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Am J Vet Res 42:888-891,1981. 17. Genetzky RM, Hagemoser WA: Physical and dinical pathological findings associated with experimentally induced rupture of the equine udnary bladder. Can Vet J 26:391-395,1985. 18. Gosset KA, French DD: Effect of age on liver enzyme activitiesin serum of healthy Quarter Horses. Am VetJRes 45:354356,1984. 19. Hjortkjaer RK, Svendsen CK: Simulated small intestinal volvulus in the anesthetized horse. Nord VetMed31:456-483,1979. 20. Hunt E, Tennant BC, Whitiock RH: Interpretation of peritoneal fluid erythrecyte counts in horses with abdominal disease. Proc 2nd Equine Colic Res Syrup Vot 2, Athens, University of Georgia, 168-174,1986. 21. Kitchen H, Rossdale PD: Metabolic profiles of newborn foals. J Reprod Fert Supp, 23:705-707,1975. 22. Kral F, LaGrange W: Colics of the horse. Published by the authors, Philadelphia, PA, 24-26,1956. 23. Mattocks AM, EI-Bassiouni EA: Peritoneal Dialysis: a review. J Pharma Sci 60:1767-1981,1971. 24. McGrath JP: Exfoliative cytology of equine peritoneal fluid- an adjunct to hematologic examination. In: Proc 1st Int Symp on Equine Hematology, ed by Am Assoc Equine Pract, 408416,1985. 25. Moore JN, Traver DS, Turner MF et al: Lactic acid concentration in peritoneal fluid of normal and diseased horses. Res Vet Sci 23:117-118,1977. 26. Morris DD, Johnson JK: Peritoneal fluid constituents in horses with colic due to small intestinal disease. In: Proc 2ndEquine Colic Res Syrup,Vol 2, Athens, University of Georgia, 134-142,1986. 27. Nelson AW, Collier JR, Griner LA et al: Acute surgical colonic infarction in the horse. Am J VetRes 29:315-327,1968. 28. Nelson AW: Analysis of equine peritoneal fluid. VetClin North Am (Large Animal Practice) 1:267-274,1979. 29. Parry BW: Use of clinical pathology in evaluation of horses with colic. Vet Clin North Am (Large Animal Practice) 179189,1985. 30. Prec 1st Int Conf on Vet Perinatology, Odando, FL, 1988. In: Perinatology, ed by Rossdale PD, Silver M, Rose J. Equine Vet J, Suppl 5,1988. 31. Richardson DW: Urogenital problems in the neonatal foal. VetClin North Am (Large Animal Practice) 179-189,1985. 32. Roberts MC: The development and distribution of mucosal enzymes in the small intestine of the fetus and young foal. J Reprod Fert, 23:717-723,1975. 33. Rose RJ, Blackhouse W, Chair W: Plasma biochemistry changes in Thoroughbred foals during the firstweek of life. JReprod Fert 27:601-605,1979. 34. Schalm OW: Significance of plasma fibrinogen in clinical disorders of the horse. In: Proc 1stInt Symp on Equine Hematology, ed by Am Assoc Equine Pract, 159-164,1975. 35. Schumacher J, Spano J, Moll D: Effects of enterocentesis on peritoneal fluid constituents in the horse. JAm Vet Med Assoc 186:1301-1303,1985. 36. Sunshine P, Herbst JJ, Koldovsky Oet al: Adaptation of the gastrointestinal tract to extrauterine life. Ann NY Acad Sci 176:16-29,1971. 37. Smyth GB: Effects of age, sex and postmortem interval on intestinal lengths of horses during development. Equine Vet J 20:104-108,1998. 38. Swanwick RA, Wilkinson JS: A clinical evaluation of abdominal paracentesis in the horse. Aust VetJ 52:109-117,1976. 39. Tulleners EP: Complications of abdominocentesis in the horse. JAm Vet Med Assoc 182:232-234,1983. 40. Vaughn JR: Digestive disturbances in the horse with intestinal obstruction. In: Proc 16th Ann Cony Am Assoc Equine Pract 343-348,1970. 41. West JE: Diagnostic cytology in the equine species: overview, effusions (peritoneal, pleural and synovial joint and transtracheal wash. In: Proc Ann Cony Am Assoc Equine Pract 169201,1984. 42. Wilson JH: gastrointestinal problems in foals. In: Current therapy in Equine Medicine, ed 2, ed by NE Robinson, Philadelphia, WB Saunders Co., 232-241,1987.
EQUINE VETERINARY SCIENCE
•
~
kJ Refereed
REFERENCEVALUESOF PERITONEALFLUIDFROMHEALTHYFOALS Eddy Behrens, MV; Maria E. Parraga, MV, MS; Alexander Nassiff, MV and Nobel DelMar, MV
SUMMARY
Thirty-two clinically healthy Thoroughbred foals, aged 14 to 75 days ~--45.5 days), were used to evaluate several parameters in peritoneal fluid. The evaluation revealed clear, pale yellow fluid with a mean total protein concentration of 1.8 gm/dl + 0.7 ~.SD). The mean total of nucleated cell count was 1,418/ul + 1,077, which was distributed as follows: neutrophils 15.3% + 20.4, large mononuclear cells 43.8% + 24.8, small mononuclear cells 22.3% + 24.5. Eosinophils were not observed in any sample and the erythrocyte count was 12,070/ul + 15,219. Nine different biochemical parameters were determined in paired samples of peritoneal fluid (PF) and serum. Results of the biochemical analysis of PF were : glucose 136.9 mg/dl + 21, aspartate aminotransferase (AST) 50 IU/ L + 18.9, alkaline phosphatase 43.6 IU/L + 15.15, lactate dehydrogenase (LDI-/)46. 5 IU/L + 22.9, creatinine 1.39 mg/ dl + 0.22, sodium 133.6 mEq/L + 2.18, chloride 98.7 mEq/ L + 4.16, potassium 4.2 mEq/L + 0.36 and fibrinogen <200400 mg/dl. When differences of these parameters for serum and PF were analyzed by paired comparisons, all were significant (P<0.05) except those for creatinine and sodium. A valuable aid for determining the probable cause of abdominal pain is analysis of peritoneal fluid (PF). Analysis of peritoneal fluid is helpful in assessing other abdominal conditions such as chronic diarrhea, parasitic larvals Author'saddress: Dr. Behrens, Large Animal Clinic, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843-4475. Acknowledgements:The authors thank Ing Rafael Branger for his financial support and Haras Las Acacias, El Bosque and Monumental for permitting us to work with their foals.
348
migrans, abdominal abscessation, granulomatous enteritis and metastatic gastric squamous cell carcinoma. This ancillary aid is even more important for foals because rectal palpation is impossible due to their small size. Abdominocentesis of the horse was described more than 30 years agoz2 and is now a routine procedure for evaluation of horses with abdominal distress. Evaluation of PF from horses with acute abdominal conditions is well documented;4,s,s,9,2°,24,a62s.~.41 however, most of the literature deals with adult horses. Although the field of equine neonatology has been recently advancing, 3° we were not able to find studies of analysis of PF from healthy foals. The serum and PF concemrations of aspartate transaminase (AST), l° alkaline phosphatase (ALP),13,16,~and lactate dehydrogenase (LDH),4° as well as the concentrations of lactic acid, 25 fibronectin, TMand phosphatea have been reported. The aim of those determinations was to search for an accurate index to predict early compromise of bowel; however, the sensitivity and specificity have been variable. ~ The purposes of this study were: 1) to report reference values for PF from healthy foals, 2) to report values of different biochemical parameters evaluated in paired samples of serum and PF and 3) to determine the relationship of those parameters between serum and PF.
MATERIALS AND METHODS
A total of 45 clinically healthy Thoroughbred foals, ranging from 14 to 75 days old ~---44.5 days old) from 3 different farms, were used in this study. The foals had not
EQUINE VETERINARYSCIENCE
Table
1.
Methods used for determination of each biochemical parameter. Test Aspartate aminotransferase (AST). Lactate dehydrcgenase (LDH) Alkaline phospharase (ALP) Glucose Creatinine Chloride thiocyanate
Conct. 0-450UV 0-1000UV 0-1200 0-400 0-10 0-130
Method Measurement of NADH/NAD Measurement of NADH/NAD P-nitrophenyl Phosphate Colorimetric glucose oxidase Colorimetric picric acid Colorimetric mercuric
Sodiumand potassiumconcentrations were measured by flame emission photometry.
been dewormed and had onlyreceived tetanus prophylaxis. The PF and blood samples were taken early in the morning before the foals were turned out to the pasture with their mares. The abdominocentesis was performed with the foal in standing position. The foals were manually restrained with 1 or 2 assistants, no chemical restraint was used. The most dependent portion of the abdomen between the exyphoid process and the umbilicus was clipped and prepared for an aseptic procedure. The operator, wearing sterile surgical gloves, introduced an 18 gauge x 2.5-cm needle into the linea alba and then slowly advanced it until fluid was obtained. If no fluid was obtained, the needle was rotated, repositioned, or aspiration with a syringe was performed. If no fluid was obtained, one additional needle was inlroduced a few centimeters from the first needle. Samples were assessed for color and coagulability. Peritoneal fluid was collected in a tube that contained ethylene diaminetetra-acetic acid (EDTA) for cytological analysis and in a tube that contained no anticoagulant for biochemical analysis. The latter sample was immediately centrifuged at 1,500 rpm for 5 minutes, and the supernatant was removed and frozen at -30°C until further analyzed. The cells in the former sample were counted with a hemocytometer. The diluting fluid for the red blood cells was Gower's solution and that for the white cell count was 2% acetic acid. Both counts were performed using a 1:20 dilution. After the cells were counted, the EDTA-containing sample was centrifuged at 1,500 rpm for 5 minutes and a smear was made from the sediment. The smear was stained with Wright's stain and a differential cell count was performed (200 cells counted). Three groups of cells were considered: neutrophils, large mononuclear cells (macrophages and/or mesothelial cells) and small mononuclear cells (lymphocytes). The estimation of total proteins in the effusion was determined by refractometry and the concentration of fibrinogen was determined by heat-precipitation at 56°C for 3 minutes. The biochemical determinations of the supernatant of PF, as well as those of serum were performed using a Boehringer Mannheim photometer. =The methods used for the determination of each biochemical parameter are listed in Table 1. =Model4010, Mannheim,W. Germany.
Volume 10, Number 5, 1990
The statistical analysis of all values except those for fibrinogen was by student's t-test for paired comparisons, b Values for fibrinogen were ordinal data and were, therefore, analyzed nonparametrically for paired comparisons. =The values were significantly different from one another at P<0.05.
RESULTS
This research initially included 45 foals, but the abdominocentesis was completed with 32 foals. Of the remaining 13 foals, PF was not obtained from seven foals, the amount of PF was insufficient from 5 foals to be analyzed, and in one foal the values obtained were indicative of an underlying disease not detected during physical examination. Therefore, the investigation was performed with 32 foals (16 females and 16 males). The cytologic evaluation is shown in Table 2. The biochemical parameters concurrently evamated in peritoneal fluid and serum were analyzed individually, the results are shown in Table 3. Values of parameters in the two samples (PF and serum) were compared and statistically analyzed with a student's t-test for paired comparison. The results are shown in Table 4. The values for fibrinogen were compared and statistically analyzed by a nonparametric method.=
DISCUSSION
Peritoneal fluid was not obtained from seven foals, which may occur in normal adult horses,a In five foals the quantity of PF was very small, in this respect the foal may have a lower rate of PF production than the adult horse in which 60 mg/hr has been reported. 27Other possible causes remain speculative but include the following: altered gravitational flow of PF (compartmentalization) by abdominal oSnedecor,GW and Cochran WG: The comparisonsof 2 samples,IN: Stalis~cal Methods, 7th edition,the Iowa State University Press,Ames, IA, 83-106,1982. cRosner B: WilcoxonSign rank test, In: Fundamentals of BiostatiMics, PWS Publishers,Boston, MA, 270-276,1984.
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Table foals.
2
Cytologic evaluation of peritoneal fluid from 32
Peritoneal fluid Red Blood Cells (/ul) Nucleated cells (/ul) Neutrophils (%) Lg. Mononuclear cells (%) Sm.Mononuclear cells (%) Eosinophils (%) Total protein (gm/dl)
Mean 12,070 1,418 15.3 43.8 22.3
SD 15,219 1,077 20.4 24.0 24.5
1.8
0.7
viscera, misalignment of the needle with a pool of PF, restricted flow of PF by adhesions, production of PF reduced by dehydration, and inadvertent insertion of needle into the falciform ligament. In any event, the volume per se' is not a reliable indicator for assessment of PF since many times a small volume of normal fluid may be obtained in horses with compromised bowel (inguinal or diaphragmatic hernias or flcocecal intussusception). These conditions are not commonly observed in foals. Conversely, a foal with a small perforated ulcer in the stomach or the duodenum, may not show abnormal changes in PF because of omental walling-off activity.42 The intestine was not punctured during the abdominocentesis procedure although a sterile disposable 18 gnage x 2.5-cm needle was used. It has been recommended that a teat cannula be used in foals because of the increased risk of unintentional enterocentesis with a needle.3g This requires local anesthesia and a small stab incision. However, complications seldom resulted from enterocentesis, 4,2s,as if the intestinal segment that was penetrated was not distended or devitalized. TM All the samples of the PF were clear, serous and strawcolored. This physical parameter can be a rough index of the number of leukocytes present, depending on the degree of turbidity3 s The results of the cytology of the PF showed nucleated cell counts (~=1,418/ul) lower than the mean values reported for the adult horse, 4,~,2sin which up to 10,000/ul is considered normal. We found only one report describing
cytology of PF in normal foals younger than one week, but no other data was provided for comparison." A total WBC count greater than 5,000/ul was reported in five neonatal foals with abdominal distress which subsequently underwent exploratory celiotomy? We suggest that a WBC count in the PF up to 3,000/ul be considered a normal value. The presence of RBCs ~ = 12,070/ul) could have been contamination of the PF during collection, intraperitoneal hemorrhage or increased capillary permeability with diapedesis of RBCs into the PF. r The RBCs in our samples were most likely secondary to collection-induced hemorrhage, despite the gross normal appearance grossly. Eosinophils were not observed in any sample, which was unexpected because none of the foals that underwent investigation had been dewormed and some degree of eosinophilia in the PF was anticipated. Eosinophilia in PF has been supportive of a tentative diagnosis of strongylosis.4 The prevalence of Parascaris supp. infestation during the foal's first months of life and the parasite's life cycle, could influence the observation mentioned. However, the lick of eosinophils in the PF does not rule out strongylosis. 41 The lymphocytes in PF were present in low numbers (~=22.3 %) similar to adult horse values,4,~,24The predominant cells in the PF (x-+=43.8%) were large mononuclear cells (marcophages and mesothelial cells). In the adult horse lower values have been reported. 4.~,2s,38Neutrophils (R=15.3%) were less predominant than in normal adult horses.4,9,24,28
The values for protein in the PF (~=1.8 gm/dl) were similar to those of the adult horse.4,Q.2sA marked variation of protein in PF was reported depending on the method used (refractometry or chemical). A lower concentration of protein was found by refractive index, r To obtain a more accurate approximation, determinations of protein were performed by refractometry with the supernatant after centrifuging the PF for 3 minutes. The peritoneal membrane is permeable to water and most solutes, allowing a dynamic equilibrium through
Table 3. Biochemical parameters concurrently evaluated in peritoneal fluid and serum. Parameter
Peritoneal Fluid ± SD (range)
Glucose (mg/dl) AST (lUlL) ALP (lUlL) LDH (IU/L) Creatinine (mg/dl) Sodium (mEq/L) Chlodde (m Eq/L) Potassium (mEq/L) Fibrinogen (mg/dl)
136.9 +21 (100-178) 5O + 18.9 (28-120) 43.6 + 15.15 (13-82) 46.5 + 22 (23-108) 1.39 + 0.22 (0.91-1.87) 133.6+2.18 (128-137) 98.7 + 4.16 (92-106) 4.2 +0.36 (3.6-4.9) < (200-400)
Serum Y ± SD (range)
89.40 + 17.80 (59-152) 185.S0 • 45.8O (8e-433) 334.70 ±102.0 (195-667) 326.50 +75.80 (179-464) 1.39 + 0.24 (0.85-1.85) 134.3+3.38 (126-141) 96.5 + 3.63 (88-103) 4.6 +0.47 (4.0-5.6) (20O-800)
x = mean, SD = standarddeviation,AST = aspartateaminotransferase ALP = alkalinephosphatase,LDH = lactatedehydrogenase
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Table
4.
Values of parameters of PF and serum samples
analyzed with a student's t-test for paired comparison. Parameter Glucose (mg/dl) AST (IU/L) ALP (IU/L) LDH (lU/L) Creatinine (mg/dl) Sodium (mEqlL) Chloride (mEq/L) Potassium (rnEq/L) Fibrinogen (mg/dl)d
Mean differencec + SD 47.50 + 30.14 -135.78 ¢ 43.03 -291.15 + 97.54 -279.90 ¢ 73.51 0.00687 ¢ 0.2634 -0.71875 ¢ 2.842 2.187¢ 3.49 -0.44437 :t: 0.4420 (range < 200-400)
P<0.05 t
=Meandifference = value in Peritoneal Fluid -value in Serum, SD = Standard deviation, * = significant, - = non significant. K)rginal data analyzed nonparametricallyfor paired comparisons (Wilcoxons Sign Rank test).
passive diffusion between blood and peritoneal fluid?3 Therefore, the PF is a dialysate of plasma and reflects any changes in the visceral and parietal peritoneum. 6,~On that basis, the concentration of diffusible elements in plasma should be very similar to those observed in PF. We measured, in paired samples of serum and PF, 9 different biochemical parameters. 1. Glucose: the mean concentration in serum was 89.4 mg/dl + 17.8 and was very similar to the ones reported for adult horses.5,9,28 Higher values have been reported for yotmgerfoals (<1 week). ~1.2~The samples were taken from our foals early in the morning, a few hours after they had nibbled the mare's feed. Blood glucose concentrations in our foals could have been affected by feeding time and alterations of light and darkness, 9 in addition to the stress related to the restraint and abdominocentesis. The mean concentration in PF was 136.9 mg/dl + 21 and was similar to values reported for adult horses,s in which the values of glucose in PF were also greater than the corresponding values in blood, s,28 2. Aspartate aminotransferase (AST): the mean concentration in serum was 185.5 IU/L + 45.8 and the concentration in PF was 50 IU/L + 18.9. These values are similar to the ones reported for adult horses in one study,28but differ from values reported in another paper. 5 We could not explain the difference. 3. Lactic dehydrogenase (LDH): the mean concenlration in serum was 326.5 IU/L + 75.8 and the concentration in PF was 46.5 IU/L + 22.9. Our values for serum are similar to the ones reported for adult horses,5,~but our values for PF were lower. 4. Alkaline phosphatase (ALP): the mean concentration in serum was 334.7 IU/L + 102 and the mean concentration in PF was 43.6 FU/L + 15.15. These values are similar to the ones reported for adult horses, s,~6but higher when compared with another paper. 28 The concentrations of these enzymes in PF were poorly correlated with those in the serum. A similar observation has been reported for the adult horse. 5 Although the enzyrnes investigated are not tissue-specific, the liver and
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intestines contain a considerable amount. On that basis it is feasible that gross deterioration or pathological lesions of intestine or liver may be necessary in order to observe an increase in the PF. It has been shown that the intestinal mucosal enzymes in the horse have characteristic patterns of development from early life to maturity.32Since production of the intestinal enzymes depends on the differentiation and maturation of the enterocytes, we hypothesize that our foals did not have fully mature enterocytes. Even more likely, the numbers of enterocytes were probably less than those in the adult horse. This assumption could be supported by recent evidence which confirms that intestinal length will keep increasing up to 1 year of age. ar A slow rate of cellular proliferation and migration of the intestinal mucosa has been reported in suckling rats and rabbits. ~ This could also apply to our suckling foals; therefore, the slower rate of turnover of the intestinal epithelium could partly explain the low enzymatic concentration in PF. It is noteworthy that in adult horses, ALP in PF was predominantlygranulocytic in origin; therefore, it was not a reliable indicator of small intestinal disease in horses. 16The same observation could be applied to the foal. Finally, the elevated concentration of enzymes in serum could be explained by the relatively high hepatic mass (as percent of body weight) and the high enzymatic activity per gram of hepatic tissue in young animals. 13,~8 5. Creatinine: the mean concentration in serum was 1.39 mg/dl + 0.24 and the mean concentration in PF was 1.39 mg/dl + 0.22. The concentration in serum was similar to the one reported for adult horses, but the concentration in PF was lower than that of adult horses. 28 Uroperitoneum in the foal due to tears in the bladder, urachus 2 or ureters, ~2and without concomitant gastrointestinal disease, will show an unremarkable analysis of PF with the exception of an increased concentration of creatinine. It has been shown that creatinine in PF is a reliable test for uroperitoneum in the horse27 In the foal a ratio of creatinine in PF to that in serum that is greater than 2:1 is highly indicative of urperitoneum)~ Our foals' values supported the mentioned statement since they showed a ratio of 1:1. 6. Sodium, potassium and chloride: the mean concentration of sodium in serum was 134.3 mEq/L + 3.38 and the concentration in PF was 133.6 mEq/L + 2.18. The mean concentration of potassium in serum was 4.6 mEq/L + 0.47 and the mean concentration in PF was 4.2 mEq/L + 0.36. The mean concentration ofchloridein serum was 96.5 mEq/ L + 3.63 and the mean concentration in PF was 98.7 mEq/ L + 4.16. These values were similar to the ones reported for adult horses 2°,28and foals serum. ~ 7. Fibrinogen: the range of concentrations in serum was 200-800 mg/dl. This range was slightly higher than that reported for adult horses.34 The range of concentrations in PF was <200-400 mg/dl. This value is similar to the ones reported (~=200 mg/dl) for adult horses with effusions of
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PF without mesenteric thromboses. 14It is noteworthy that for 65% of the group subjected to this investigation, the concentrations of fibrinogen in the PF were less than 200 mg/ dl. Therefore, the majority of the foals had concentrations of fibrinogen in the PF that were either to minute to be detected or were absent. Since fibrinogen plays an important role in any inflammatory process through its conversion to fibrin, and its increased concentrations will decrease to normal as inflammation subsides, it might be a reliable test in abdominal disturbances. To conclude, when the biochemical parameters of paired peritoneal fluid and serum samples were compared and statistically analyzed, the enzymatic activity (AST, IDH, ALP) the concentration of glucose, potassium, chloride and fibrinogen were significantly different. Concentrations of creatinine and sodium were not significantly different.
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