of hepatichepatic disrof hepatic

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functional ai

failure), bacteria (Ieptospirosis), vascular abnormalities or lack of(portosystemic shunt, hepatic cysts), virus (infectious hepatitis portosystenvirus) and metabolic disorders (diabetes m ellitus, As arhyperadrenocorticism and hypothyroidism)":". HE in dogs

Owing to the great storage capacity of the liver and taken carefithe multiple functions performed by this organ, the early 2-hourpost]diagnosis of hepatobiliary diseases is still a challenge for safer.c1inicians being usually established when c1inical signs as F~r eascites and jaundice are noticed, because of extensive loss enzymatic r

Bm2. 1. ver Res. anim. Sci.,sa.. Paulo, v. 36, 11. J, r. 28-33, J 999.

Evaluation of preprandial and postprandial serum bileacids and plasma ammonia concentrations in heaIthydogs, and the effects of frozen storage on plasma ammoniaconcentrations'

CORRESPONDENCE TO:Márcia Mery KogikaDepartamento de CirurgiaFaculdade de Medicina Veteriná-ria e Zootecnia da USPCidade Universitária Armando deSalles Oliveira,Av. ar/ando Marques de Paiva, 87.05508-000 - São Paulo - SP.e-mail: mmkogika@usp.br

Avaliações dos níveis pré e pós-prandiais de ácidos biliaresséricos e de amônia plasmática em cães hígidos e o efeito dotempo de congelamento do plasma nas concentrações de amônia *

l-Departamento de ClínicaMédica da Faculdade deMedicina Veterinária e ZootecniadaUSP-SP

Márcia Mery KOGIKAlj Shinobu MATSUURA1j Mitika Kuribayashi HAGIWARAlj Regina Mieko Sakata MIRANDOLAljEnrico Lippi ORTOLANP

SUMMARY

Preprandial and postprandial (2 and 4 hours) serum bile acids (SBA) and pre and postprandial (2 hours) plasma ammoniaconcentrations were evaluated. Additionally, the effects of freezing (for 24 and 48 hours at -200e) were observed onplasma ammonia concentrations in 22 healthy dogs. The preprandial SBA concentration was 2.1 ± 0.3 mmol/I and 7.5 ±1.2 mmol/I and 7.8 ± IA mmol/I for samples obtained 2 and 4 hours after feeding, respectively. Fasting and postprandial(2 hours) plasma ammonia concentrations were significantly different when measurement was performed within 30minutes after blood collection (118.2 ± 13.2 mg/dl or 67.3 ± 7.5 mmol/I and 227.9 ± 59.2 mg/dl or 129.9 ± 33.7 mmol/I), but the difference between pre and postprandial concentrations was not observed when ammonia was measured insamples stored (_20°) for 24 and 48 hours. Plasma freezing makes ammonia concentrations fali considerably when theselevels were initially toa high, mainly in postprandial samples. From these results it may be suggested that canine plasmacannot be stored for later ammonia determination by using freezing as the sole stabilizer, and for SBA determinations,blood samples might be collected 2 or 4 hours after Ieeding. Plasma ammonia values obtained in this study should allowcomparisons to data obtained from dogs with hepatic disease or hepatoencephalopathy, so as to confirm the importanceof its use as means of diagnosis and prognosis in future.

UNITERMS: Bile acids; Ammonia; Dogs.

INTRODUCTION

Hepatobiliary diseases, which can develop either asacute or chronic progressive hepatic diseases, arefrequently diagnosed in dogs and cats. The most

important etiologic agents involved are toxic substances(drugs, mycotoxins, heavy metais - for example hepatitisassociated with copper in Bedlington terriers andDobermans), ischemy (anaemia and congestive right heart

"This study was supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, proc. 95/9417-1 and 95/9415-9)

28

KOGIKA,Macids andanimo Sei

'Commercial (

KOGIKA, M,M,; MATSUURA, S,; HAGIWARA, M,K,; MIRANDOLA, R,M,S,; ORTOLANI. EL Evaluation of prcprandial and postprandial serum bileacids and plasma arnrnonia concentrations in healthy dogs, and the effects of írozen storage on plasma ammonia concentrations. Braz. J. veto Res.animo Sei., São Paulo, v. 36, n. I, p. 28-33, 1999,

of hepatic parenchyma. Other symptoms are not specific ofhepatic diseases, thus making even more difficult the diagnosisof hepatic lesion or dysfunction 7.

Laboratorial assays are the most important proceduresto detect liver diseases. Nevertheless, as some dogs showminimal or none alterations in routinely used hepatobiliaryscreening tests, other tests such as serum bile acids (SBA)and plasma ammonia determinations had been suggested tohelp clinicians to evaluate liver function!'.

Compared to other more traditional assays, serum bileacids determination can be considered one of the mostimportant tests for the diagnosis of hepatobiliary disease,because of the high sensitivity and specificity. Bile acids aresynthesized in liver from cholesterol, being later stored ingallbladder and released into the small intestinepostprandially 1.9.10.25.

Although it has been suggested that only postprandialSBA determination should be considered in the diagnosis ofhepatobiliary diseases in dogsI3.25, some authors recommendtheir determination in both, fasting and 2-hour postprandialsamplesX,9,IO,IX,19.

According to Center? and Meyer et aUI, plasmaammonia concentration is as important as SBA determinationto detect hepatic dysfunction. Ammonia comes from proteindegradation in colon, being considered one of the substancesresponsible for hepatic encephalopathy (HE) manifestation,a clinical syndrome characterized by abnormal mental statusand impaired neurological function in animais that haveadvanced liver disease (acute or chronic) and/or severeportosystemic shunts=':".

Due to great variability of plasma ammoniaconcentrations in dogs, even in fasting samples, manyresearchers recommended the ammonia tolerance test inwhich the plasma concentration ofthe substance is measuredbefore and after administration of ammonium chloride (NH4CI,100 mg/kg) via stomach tube''!':". Slightly increased post-challenge blood ammonia levels are considered normal inhealthy dogs, but markedly increased may indicate decreasedfunctional ability owing to the reduction ofhepatic parenchymaar lack of integrity of the portal blood supply due topartosystemic shunts.

As ammonia chloride may induce or enhance signs ofHE in dogs, even in asymptomatic ones, the test should betaken carefully or, according to Dial", should be replaced by2-hour postprandial plasma ammonia determination, which issafer.

For evaluation of blood ammonia concentration, theenzymatic method has been routinely used, but now an easy

ná-

de

87.

icadenia

iesitisvs,

.ndrlyforasss

'Commercial canned Iood (Pcdigree-chicken flavour), 250 gmll5 kg BW (supplied by ÉfTcm Produtos Alimentícios/Waltham).

29

and feasible method, using a selective ion electrode isavailable". It allows determining high levels of circulatingammonia (up to 100,000 mg/dl or 1,000 ppm) without the needof diluting the sample, making the method an ideal one foruse in clinical routine.

Considering the high instability of ammonia, it isrecommended to do its determination in fresh plasma within30 minutes after blood collection'v'"; although, according tosome researchersV':", the plasma could be stored at -20°Cfor I to 2 days without any alteration in ammonia concentration.

In this paper, the authcrs present the pre andpostprandial values of SBA and ammonia, the latter measuredby means of ion selective electrode method in healthy dogs.Beside this, the influence offrczen storage on ammonia levelsof the sample, as well as the optimurn postprandial time forblood collection for SBA analysis were studied.

MATERIAL AND METHOD

Dogs - Twenty-two healthy mixed-breed dogs, bothsexes, aged 2 to 6 years, from Experimental Animal HoldingFacility of Departamento de Clínica Médica 1 Faculdade deMedicina Veterinária e Zootecnia da Universidade de SãoPaulo or housed dogs were used for this study. Health wasassessed by history, clinical examination and confirmed byroutine biochemicalliverfunctions tests (alanine transaminase,alkaline phosphatase, total protein and alburnin).

Samples - Blood was collected, via venipuncture, fromeach dog after a 12-hour fast and 2 hours postprandially",another postprandial sample (4 hours after feeding) wasobtained for serum bile acids determination. Serum for bileacids determination was separated immediately after bloodcollection and stored at -20°C.

For ammonia measurement, blood was collected intoheparinized glass vacuum tubes, fasting and 2-hourpostprandial samples, centrifuged at 2,000g for 10 minutes,and the plasma separate d (3 aliquots) and ammoniadetermination made within 30 minutes; two other aliquots wereimmediately stored at -20°C.

Serum bile acids (SBA) determination - Serum bileacids concentrations were obtained by use of commercialcolorimetric enzymatic method - Enzibile=Prod. n°. 10026821Standards Prod. n°. 1002657 (NYCOMED PHARMA AS).

Plasma ammonia determination - Plasma ammoniaconcentration was determined in fresh plasma sample (kept

KOGIKA, M.M.; MATSUURA, S.; HAGIWARA, M.K.; MIRANDOLA, R.M.S.; ORTOLANI, E.L. Evaluation ofpreprandial and postprandial serum bileacids and plasma ammonia conccntrations in healthy dogs, and the effects of frozen storage on plasma ammonia concentrations. Braz. J. veto Res.animo Sei., São Paulo, v. 36, n. I, p. 28-33, 1999.

in ice bath) obtained within 30 minutes after blood collection,and in samples stored at -20°C for 24 and 48 hours, fastingand postprandial blood samples, using a specific ion electrodemethod, as referred by Attili et ai. Ammonia was measuredby use of Orion" electrode - model 95-12; briefly, I ml ofplasma sample was diluted with 2 ml of pH 12 buffer solution(Titrisol n°. 9892 Merck), homogenized with magnetic stirrer,and the m V reading were measured with SA 720 Meter(Procyon). Standard solutions (0.1 to 1,000 ppm of ammonia)were diluted with pH 10.5 buffer solution (8.1 parts of pH 10buffer Titrisol n°. 9890 + 1.9 parts of pH 12 buffer Titrisol n°.9892), processed likewise and the results in mV were plottedin monolog paper. Ammonia concentration was ca\culatedaccording to standard logarithmic curve referred above.

Statistical analysis - To assess whether there is adifference between pre and postprandial SBA concentrations,and also 2 and 4 hours after feeding SBA values, pairedStudent t test was conducted at p<0.05**. Analysis of variance(ANOVA) was used to evaluate difference between plasmaammonia concentrations obtained 30 minutes aftervenipuncture (controls) and aliquots stored at -20°C for 24and 48 hours. This test was followed by Dunnett MultipleTest, and significance was considered at a leveI of a = 5%.In order to detect a difference between pre and postprandialplasma ammonia levels, paired Student t test was alsoconducted at p<0.05.

RESULTS

Mean and standard error mean values of SBAconcentration were 2.1 ± 0.3 mmol/l (preprandial), 7.5 ± 1.2mmol/I and 7.8 ± 1.4 mmol/I (postprandial, for samplesobtained 2 and 4 hours after food ingestion, respectively)(Tab.I). Analysis of data showed significant differencebetween mean pre and postprandial SBA concentrations.However, no difference was detected between postprandialSBA values (2 and 4 hours after feeding).

Fasting and postprandial plasma ammoniaconcentrations that were measured within 30 minutes aftervenipuncture are presented in Tab. 2. Fasting plasmaammonia concentrations ranged frorn 74 to 350 mg/dl (118 ±

13.2 mg/dl) and rase to values ranging 60 to I, I00 mg/dl (227.9± 59.2 mg/dl) for samples collected 2 hours after feeding,with significant difference between pre and postprandialplasma ammonia concentrations.

"Graph Pad INSTAT TM, Y.2.0I, Copyright 1990 - 1993.

30

Prepwere signifsimilar resiand Jensen(0.3 mmol/(0.3 mol/l te

SD = standard deviation; bile acids aiSEM = standard errar mean; digestive p* = significantly different frorn the postprandial (2 and 4-hr) mean values B k(p<0.05, paired t test). ec ett el

postprandis

Pre and postprandial plasma ammonia concentrations, after feedirafter plasma storage at -20°C for 24 and 48 hours, are also . In 01

showed in Tab. 2. There was a significant difference between obtained fn

d d· I . h was not 01pre an postpran Ia concentrations w en measurement was .f d 30' f . b diff convention.per orme rrunutes a ter verupuncture, ut no I rerence .

consideredwas observed when samples were stored for 24 and 48 hours, J IX

h . h I 20°C k . ensen obs owmg t at pasma storage, even at - ,ma es arnrnorua bconcentrations fall considerably when these levels are high. etween 2 ;

300r-------~~==================~Ic::J Preprandial •• postprandiall

250

200

~ 150;:s

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KOGIKA, Macids andanimo Sei

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StorageValuesMeanSDSEMMaximumMinimum30min 24 hr 48 hr

TIMESD = standarSEM = stand• Significant (30 minutes a•• Significant24 and 48 h·To convert m

Fig. 1 showconcentrati

Figure 1Variations in plasma ammonia concentrations (preprandial andpostprandial samples) determined within 30 minutes aftercollection and in samples stored for 24 and 48 hours (-20°C) fromhealthy dogs.

Table 1Pre and postprandial (2 and 4-hr) serum bile acids concentrations(mmol/I) in healthy dogs. São Paulo, 1997.

Bile Acids (mmol/I)

Preprandial 2-hr Postprandial 4-hr Postprandial

Valuesmean 2.1"

1.50.36.20.3

7.55.51.221.20.3

7.86.51.4

22.81.9

SDSEMmaximunminimun

»le KOGIKA, M.M.; MATSUURA, S.; HAGIWARA, M.K.; MIRANDOLA, R.M.S.; ORTOLANI, E.L. Evaluation of preprandial and postprandial serum bileteso acids and plasma ammonia concentrations in healthy dogs, and lhe effects of frozen storagc on plasma ammonia concentrations. Braz. J. veto Res.

animo Sei., São Paulo, v. 36, n. I, p. 28-33, 1999.

Table2Pre and postprandial plasma ammonia concentrations (mg/dl) insamples determined within 30 minutes after blood collection and insamples stored for 24 and 48 hour at -20°C. São Paulo, 1997.

Plasma ammonia (lJg/dl)

StaragePreprandial Postprandial

30 min 24hr 48hr 30 min 24hr 48hrValuesMeanSOSEMMaximumMinimum

118.2' 119.0 139.362.2 40.8 44.013.2 8.7 9.4350 210 24074 60 62

227.9"277.759.2110060

111.130.06.416060

127.937.88.020566

andfter'om

SD = standard deviation;SEM = standard errar mean;"Significam difference from the postprandial mean value obtained within30 minutes after collection (p<0.05, paired t test);•• Significantly different frorn the means observed in samples stored ror24 and 48 h -postprandial (ANOVA - Dunnett Multiple Test, a = 50/0).To convert mg/dl to mmol/I, multiply by 0.575

ons

Fig. 1 shows the mean pre and postprandial plasma ammoniaconcentrations.

dial DISCUSSION

Preprandial SBA concentrations found in this studywere significantly different from postprandial SBA values;similar results had been described by Counsell; Lumsderi"and Jensen IX. Postprandial SBA concentrations varied more(0.3 mmol/I to 21 .2 mmol/l) than preprandial concentrations(0.3 moI/I to 6.2 mmol/l), which could be explained by differentbile acids absorption peaks and quantity offood ingested anddigestive process in genera)3,23.26. Due to these variations,Beckett et al,' and Washizu et al:" recommend thatpostprandial determination should be made within 8 hoursafter feeding.

In our study significant difference between valuesobtained from samples collected 2 and 4 hours after feedingwas not observed, allowing to the conclusion that theconventional collecting time (2 hours after feeding) might beconsidered suitable for routine bile acids determination.Jensen" observed similar results with determinations madebetween 2 and 6 hours after feeding.

ilues

ons,also.eenwasmcemrs,amaugh.

Preprandial plasma ammonia concentrations, obtainedin fresh samples and determined by specific ion electrodemethod, were similar to those described by Hardy"; Bunch"and Willard; Twedr". Postprandial values were significantlydifferent from preprandial ones, but now because of thepaucity of information in the literature and the high variabilityamong the samples collected after 2 hours of the meal, themeaning and usefulness of pre and postprandial sampling forammonia determination are still to be c\eared.

Due to plasma amrnonia instability, the authorsrecommended its determination within 30 minutes aftervenipuncture+v'"?", although other researchers did notobserve difference in values obtained from plasma stored at-20°C until 48 hours after venipuncture+Vv".

Concerning canine species, data obtained in this studydemonstrated that plasma storage at -20°C did not modifypreprandial low plasma ammonia values measured 30minutes, 24 and 48 hours after venipuncture. However, as topostprandial ammonia levels from samples stored at -20°Cfor 24 and 48 hours there was a significant difference amongvalues obtained in the same samples, when they wereevaluated at 30 minutes, 24 and 48 hours after venipuncture.Same results had been related by Hitt; Jones'? and Dial",which explained that ammonia concentrations might decreaseduring storage due to vaporous loss as equilibrium is establishedbetween aqueous and gaseous phases. These results suggestthat measurement should be made within 30 minutes afterblood collection, at least when plasma levels are elevated.

Therefore, it can be recommended that, for SBAdeterminations, blood samples should be collected 2 or 4 hoursafter feeding and, for plasma ammonia determinations,measurement should be made as soon as possible aftervenipuncture (within 30 minutes).

Regarding to pre and postprandial plasma ammoniadeterminations, values obtained in this study should becompared to those obtained in animaIs with hepatic diseaseor HE, so as to confirm the importance of its use as meansof diagnosis in future.

CONCLUSIONS

- Postprandial SBA evaluation can be performedindifferently 2 01' 4 hours after feeding;

- Plasma ammonia determination should be made within30 minutes after venipuncture.

31

KOGIKA, M.M.; MATSUURA, s.; HAGIWARA, M.K.; MIRANDOLA, R.M.S.; ORTOLANI, E.L. Evaluation of preprandial and postprandial serum bileacids and plasma ammonia concentrations in healthy dogs, and the effects of frozen storage on plasma ammonia concentrations. Braz . .T. veto Res.animo Sei., São Paulo, v. 36, n. I, p. 28·33, 1999.

KOGIKA,bile aciRes. an

RESUMO

Foram avaliadas as concentrações pré e pós-prandiais de ácidos biliares séricos (2 e 4 horas) e de amônia plasmática(2 horas) em vinte e dois cães hígidos. O efeito do tempo de armazenamento (à temperatura de -20°C) do plasmasobre as concentrações de amônia também foi estudado. A média e o erro padrão da média em relação aos valorespré-prandias de ácidos biliares séricos (ABS) foram de 2,1 ± 0,3 mmol/l e de 7,5 ± 1,2 momol/I e 7,8 ± 1,4 mmol/I para os valores pós-prandiais de 2 e 4 horas, respectivamente. As concentrações plasmáticas de amônia pré epós-prandias (118,2 ± 13,2 mg/dl ou 67,3 ± 7,5 mmol/l e 227,9 ± 59,2 mg/dl ou 129,9 ± 33,7 mmol/l), diferiram(p<0,05) nas amostras mensuradas em até 30 minutos após a colheita de sangue; entretanto, a diferença entre osvalores pré e pós-prandiais deixou de existir quando a amônia era mensurada nas amostras que foram congeladaspelo período de 24 e 48 horas. Observou-se que os valores de amônia das amostras pós-prandiais, que foramcongeladas, apresentavam-se mais baixos quando comparados aos valores obtidos da mesma amostra mensuradaem até 30 minutos após a colheita, e a diminuição da concentração de amônia era mais drástica quando os valoresiniciais eram muito elevados. Os resultados obtidos sugerem que o plasma do cão não pode ser estocado paraposterior determinação de amônia, utilizando-se apenas do congelamento como forma de estabilizar a amônia.Para a avaliação dos valores séricos pós-prandiais de ácidos biliares, sugere-se que a colheita de sangue possa serefetuada em 2 ou 4 horas após a alimentação. Os valores de amônia plasmática obtidos no presente estudo podempermitir a comparação com os valores observados em cães com doença hepática ou encefalopatia e assim confirmara importância da sua utilização no diagnóstico e prognóstico.

UNITERMOS: Ácidos biliares; Amônia; Cães.

19- JENSbil,JOI

20- JENSIMI

21- MEYBRhYIRe

22- OGILpianinVel

23- SHAReicir:p.7

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9- CENTER, S.A.; BALDWIN, B.H.; ERB, H.N.; TENNANT, B.e. Bileacid concentrations in the diagnosis of hepatobiliary disease inthe dog. Journal of American Veterinary Medical Association,v.187, n.9, p.935-40, 1985.

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n bile KOGlKA,M.M.; MATSUURA, S.; HAGIWARA, M.K.; MIRANDOLA, R.M.S.; ORTOLANI, E.L. Evaluation of preprandial and postprandial serumRes. bile acids and plasma ammonia concentrations in healthy dogs, and the effects of frozen storage on plasma ammonia concentrations. Braz. J. veto

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NTZ,-hou rsis ofinary

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Thectice,

ids in

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ACH,1985-ation,

I timeurnal

serum, v.38,

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Received: 01/04/1998Accepted: 22/07/1998