:n In: CraBfold, R, D.á13ersion and mortalily of.j ,{rch Geflügelk 62,

1! deposition iD broiIor

:erlsiics of the broiler

]idr w F, (l99])| As-::naging and spectros-

:uc!lon World Animal

: lhe estimation of the

ol gro$,th in broi]ersl.r PIoduction', celle,

]me one, second Edi-

: iraits. abdominal fat.ien during a twenty

::1ation of breast mus_:_2ing (MRI) Proc 9

son ol breast mllsclelence imagin8 (MRI)Jlt^ \íeat, Zaragoza

Acla l/elerinaria Flungalica 4ó (2), pp 1.15 ]56(]998)

TrIYROID VOLUMETRJC MEASUREMENTAND QUANTITATIVE TIIYROID SCINTIGRAPI{Y IN DOGS

L. BALoGHI, Julianna THURócZv2, L Brrst3, Margit KulcsÁn2, Gv. A, JÁNoKll,P. Ruoasa and Gy. HuszeNtcze2

lDepartment ofApplied Radioisotopes, National 'Frederic Joliot-Curie' ResearchInstifute fol Radiobiology and Radiohygiene, H-l775 Budapest, P,O. Box 10l, Hungary;

2Department of Obstetrics and Gynaeco)ogy. ]Depanment of Pathology, andaDepartment ofPhysiology and Biochemistry, Univelsity ofVeterinary science,

H-l400 Budapest, P.O. Box 2, Hungary

(Received Septembet 19,1997; accepted January 6, 1998)

Thyíoid volumetlic measulement combined with quantitative'9'Tc-per-technetate th)Toid scintigraphy was performed in 62 clinical canine patients hav-ing suspected thyroid abnormalities, Euthyroid dogs (n : 22) had a total thyroidsize of 3.60 + 1,36 cm3, the thyroid/salivary gland region of intelest (RoI) ratiowas 2.01 } 0.55, the thyroidóackground ROI ratio was 3,8ó,l 0.90, and 20-minthyroid radioactivity uptake was 1,17 t 0,7l% ofthe injected dose (I. D,). ByStudent's unpaired test, thyroid size of the hypoüyroid group (n = 36) was notstatistically different from that of the euthyroid dogs, but all other quantitatiYedata (e.g. thyroiüsalivary gland ROI ratio: 1.08 + 0.56, üyroid,/backgrorrnd ROIrutio = 2.32 t 0.'70, and 20-min thyroid ladioactivity uptake : 0.34 + 0.22y,J of theL D.) wele significantly (p < 0,00l) lower in hypothyroid than in euthyroid dogs.Evaluating the aboveJisted quantitative data of separated thyroid lobes by Stu-dent's paired test, there was no significant diffelence between the left and the rightlobe eithel in the euthyroid or in the hypothyroid group.

Key words: 99'nTc-peftechnetate quantitative thyroid scintiglaphy, thyroidsize evaluation, volumetric measulements, TRH stimulation test, skin biopsy,euth}.roid, hypothyroid, hyperthyroid, thyroid malignancies, dog

Thyroid abnormalities are the most commonly diagnosed endocrine disor-ders in dogs (Paradis et aI., 1991; Chastain and Panciera, 1995). The clinicalsymptoms of thyroid diseases are numerous, variable, of low specificity, andrarely pathognomonic; biochemical examinations (total cholesterol, TCh; cre-atine phosphokinase, CK; complete blood cell count, CBC) can support tlre cli-nician's suspicion and provide additional data, but the final diagnosis cannot bereached without thyroid hormone (thyroxine, Ta; triiodothyronine, T3) measure-ments. Thyroid scintigraphy is also widely used in both human and veterinarymedicine, because by other diagnostic imaging techniques (X-ray, ultrasonogra-

0236-6290/98/s 5 00 @ 1998 Akadémiai Kiadó, Budapes!

146 BAI-oGH et al

phy) thyroid imaging is often difficult, needs expensive instrumentation and anexperienced examiner but yields only morphological data.

All investigators agree with the primary need of plasma thyroid hormonemeasurements in canine tlryroid diseases. Some endocrinologists, however, statethat the biologically active hormone (T3) levels inadequately represent thyroidgland function (Jeffers, 1990). The basis of this statement is that, unlike Tawhich is produced entirely by the thyroid gland, 40-80% of T3 is produced fromthe peripheral conversion of T4 to T3. The remaining pottion of T3 is producedby the thyroid gland. T3 is produced at the site of its action, i.e. predominantlyilrtracellularly, so its activity may not be accurate|y reflected by serum levels,Fufthermore, triiodotliyronine levels in the plasma often decrease on|y at a latestage of hypothyroidism, and liver diseases, certain drugs etc. can sometimes giverise to false diagnosis because oftheir anti-T3 effects (Jeffers, l990).

To distinguish between primary (thyroid) and secondary (hypophyseal)hypothyroidism, the TSH response test is widely used because of its well-recognised clinical usefulness (OIiver and Held, l985; Jeffers, 1990; Paradis eta|., 1991; Hammerling et al., 1992; Chastain and Panciera, l995; Rijnberk,l996), Although the TRH stimulation test is known to be a less consistent tool(Paradis et al., 1991; Frank, l996), a few investigators have reported the advan-tage of its use in diagnosing primary, secondary and tertiary hypothyroidism(Lothrop et al,, 1984; Evinger et al., 1985; Rudas et a|,, |994a; Grunau et al,,l995). Furthermore, TRH is less expensive than TSH. TRH is a tripeptide arrdits structure is the same in all mammalian species, so it may be used in compan-ion as well as in large animals,

Histopathological evaluation of skirr biopsies is an acknowledged methodin the diagnostic work-up ofcanine hypothyroidism (Gross et al., l992; Rudas etal., |994b; Scott et al., 1995), Although it is not regarded as a definitive metlrodfor determining the thyroid status of an animal, it can serve as a surrogate test insupporting the findings of other investigations. Diseases with atrophic patternincluded in the differential diagnosis of hypothyroidism are hyperadrenocorti-cism, castration-responsive dermatosis/growth hormone responsive dermatosis,female hyperoestrogenism, and Sertoli-cell tumour in the ma|e (Gross et al,,l992; Rudas et a|., l994b; Scott et al., l995). Few lristopathological changes aresuggestive of hypothyroidism: acanthosis and hyperplasia of the dermis and fol-licular infundibulum in the face of follicular groMh cycle arrest, eosinophilicvacuolisation of arrector pili muscles, and myxoedema of the dermis, althoughthe last two lesions are not observed in all cases (Gross et al., 1992).

Three radionuclides 1I23l-iodine,'''I-iodine and 99'Technetium pertech-netate (99"'Tco4 ) aIe potentially available for thyroid imaging. The choice of theideal isotope is governed by various factors including instrumental facilities, ra-diation protection aspects, goals ofexamination and, ofcourse, budgetary limits.

1cla yeletiharia Hungarica 46, ]998

:re two iodine isotopes se;.: they are not only concen:,-rporated into thyroid hc,:

aj or disadvantages, Beca-.rd P-particle emission. ]

]

.:d due to its hard gamma,etor. l23I-iodine would be

::mma energy (l56 KeY ll :

:_rt unfortunately it is te-,,o ::reld uct). Currently 99'"TcO.

. _.id scanning because of it:;.. p-particles, Tn = 6 h) ar..

: does not organificate in::apped in the thyroid gland

Static thyroid scannIr::1ost common scintigraph i

,98ó; Benard and Regnier.99.1; Brawner, 1996). Th:_ess studied; still, there are

:nd calculated ROI ratios l

Sjollema et al., l989; Nap ,

:ats (Beck et al,, 1985) TL

ar ailable on quantitative th:ion tests in dogs. Th;roidnethod has by now beconre:l.. 1994), but thyroid siztunique even in the field e.i,been publislred on tl]e use .1996).

,1ninaIs

Sixty{wo canitre pali,tbr scintigraphic exarninati..Hungary. Suspiciorr of thr r,:

cal signs (overweight or rr,malities, lethargy, palpableCBC). The age, sex, rveieht

lan

onetate,o id

T4

THYROID SCINTICRAPHY lN DOCS

_ re two iodine isotopes seem to be the most specific and most suitable nuclides,:-l rhey are not only concentrated in and trapped by tlre thyroid but are also in-:.rrporated into thyroid hormones (organification). unfortunately, both lraverajor disadvantages. Because of its longer physical half-life (T,7, : 8.06 days):nd B-particle emission,r]|I has significantly higher patient and examiner dose,:nd due to its hard gamma radiation (360 KeV) also needs a high-energy collinator, l23l-iodine would be the best thyroid scanning agent owing to its ideal

:alnma energy (l56 KeV with no P-particles) and physical half-life (T,7. = 13 h),

]t!t unfortunately it is too expensive for veterinary clinical purposes (cyclotron:roduct). Currently "'nTcoo- is the most widely used radiopharmaceutical in thy-:oid scanning because of its ideal physical properties (l40 KeV gamma-emitter,

ro P-particles, Ty,: 6 h) and relatively low price (generator product). However,rt does not organificate into thyroid hormones; it is only concentrated and:rapped in the thyroid gland.

Static thyroid scanning within l h after 99'TcOo- injection is one of tlremost common scintigraphic applications in veterinary practice (Hightower,1986; Benard and Regnier, 1989; Kintzer and Peterson, 1991; Marks et al.,1994; Brawner, l996). The dynamic aspects of thyroid scintigraphy have beeniess studied; still, there are a few publicatiorrs on thyroid radionuclide uptakeand calculated ROI ratios (thyroid/salivary gland, thyroid/background) in dogslSjollema et al,, 1989; Nap et al,, l994; Brawner, 1996; Adams et al., l997) andcats (Beck et al., 1985). To the best of our knowledge, no publication is yetavailable on quantitative thyroid scintigraphy, skin biopsies and TRH stimula-rion tests in dogs. Thyroid volumetric measurement using an ultrasonographicrnethod has by now become a more or less routine clinical procedure (Wisner etal,, 1994), but thyroid size evaluation in dogs by scintigraphic technique isunique even in the field of veterinary nuclear medicine: orrly a single paper has

been published on the use of this technique in hyperthyroid cats (Forrest et al.,]996).

Materials and methods

.1nimals

Sixty-two canine patients with suspected thyroid disorders were presentedfor scintigraphic examination at the University of Veterinary Science, Budapest,Hungary. Suspicion of thyroid involvement was based on medical history, clini-cal signs (overweight or weight loss, muscular weakness, hair and skin abnor-malities, lethargy, palpable thyroid masses) and laboratory findings (TCh, CK,CBC). The age, sex, weight and breed ofthe animals are shown in Table 1.

-om

cedrtiye ls.late

live

:al)ell-iet:rk,

ooIan-

smaL,:nd

l1,1-

odet

odinrnti-is,l.,rerl-

ic

;ir

-l-

le1-

/lcfu lakrhlarp Hunganca 1ó !,j9i

BALoGH et al

Table 1

Data ofthe Patients

Eulhyíold0r:22)

Hypothyroid(n = 36)

Hyperthyroid(n=2)

Malignancies(n=2)

Age (years) 2_14 1.5_12 6, 4.5 8, l0

Sex(female/spayed)(male/castrated)

l0/47ll

24/56lI 1/l

Body weight (kg) l142 7,5_80 5.4, 1,7

Mongrel (6)German shep-herd (5)Doberman (4)Dalmatian (2)BoxerPuliFox terrierschnauzerPinscher

DachshundDoberman

schnauzerMongrel

Pu|i (6),

Mongrel (6)Schnauzer (5)Doberman (3)Boxer (3)German shepherd (3)Poodle (2)Pug (2)Great Dane (2)Chow Chow (2)West HighlandKomondor

Skin biopsy

Skin biopsies from s r-,

':iopsy device from tlre dors;

: igls of excessive secondary

:ormaldehyde solutiol1. e]]]b.

l1atoxylin and eosin, All t,i.-

sions compatible witl] endta.:

Sc itt t i graphi c examin a t ioD j

The dogs were sedat:

:ide - xylazine combinat.

Ronrpun, Bayer, Get,tnanr

an imals were positioned ii:

.entre of the cervical regicr,

nous vein. A |arge field.,f.ras used with a generai par

tjon the syringe was counpertechnetate (Sorin Bionle,bolus and a dynamic studrlrere col|ected (128 x 1]S

study static pictures \\eIe l

using 500,000 cts prerequ ]:

catheter weTe counted (pos:.

Data were evaluated

\íediso, Budapest, Hung3:rras calculated as the per..doSe counts) evaluatil]g lh<

Ihe dynamic study. RolS 1,.

slands and backgroLttld re:

Thyroid sizes were eslil],]:

program (macro).

síalislics

Student's paired r-tetive scintigraphic data r,-,i .

thyroid groups. Studenr'_.

ttrences between tl]e eui.number of cases the hl pertistically.

TRH stimulation test

The diagnosis of hyperthyroidism was made on the basis of plasma basalT4 coDcentration measurement as described by Bellshaw and Rijnberk (1979)and resistance to stimulation by TRH (Rudas et aI., I994a). Briefly: basal sam-ple collection was carried out fíom the radial vein between 8:00 and l0:00 a.m.after tlie dogs lrad fasted for 12 h. Further samples were collected 4 and 8 h afterintravenous administration of TRH in doses of 200 pgldog. TRH (Sigma, Ger-many) used for the stimulation test was diluted in sterile physiological saline at200 pglmL concentration by our own laboratory, One or two mL blood sampleswere taken into heparinised glass syringes, and after centrifugation (1000 g,5 min) the plasma samples were stored at -l8 "C until the measurement. Thy-roxine levels were measured using a modified human radioinrmunoassay kit(Institute for Isotopes Ltd,, Budapest) which had been validated for canine Tameasurement. The criteria for euthyroid status wele a basal T1 serum level be-trveen 20 and 50 nmol,/L and an at least 50% increase in plasma Ta concentration4 h after TRH administration.

Ácta l'elerinalia Hungallca 16, 1998

TllYROID sCtNTIGfu\PllY IN DoGs l49

.Skin biopsy

Skin biopsies from suspected hypothyroid cases were taken witlr a punch'riopsy device from the dorsa] region of excessive alopecia, avoiding areas withsrgns of excessive secondary pyoderma, Samples were placed into 8% buffe.ed:onnaldehyde so|ution, embedded irr paraffin, cut at 4 pm and stained rvith hae-rlatoxylin and eosin, All biopsy samples were evaluated for the presence of le-sions conrpatible with endocrine dermatoses by the same person.

S c ítt t igrap h ic exam inotion s

The dogs were sedated by intravenous injection of ketamine hydrochlo-:ide - xylazine combination (SBH Ketamine, SelBruHa Ltd., Huirgary

-Rotnpun, Bayer, Germany) in a dose of l0 mg-l mg/kg of body weiglrt. The:ninrals were positioned irr dorsal recunrbency, the detector rvas placed at the:entre of the cervical t.egion, and a venous catheter was inserted inio the saplre-roLts vein. A large field of view analogue gamma camera (Picker Dyna, 9Ól0)iras used with a general parallel hole collimator, lmmediately before the injec-:ion the syringe was counted (predose counts), Eighty to 150 MBq 99;Tc-]eftec|]netate (Sorin Biomedica generator) was injected in less than 1 mL as aiolus and a dynamic study was begun simultarreously, Sixty 2O-second franresret,e collected (l28 x l28 x ó4) between minutes 0 and 20. After the dynamic.tt,dy static pictures were taken from dorsal and left lateral view of tlre neck,,tsing 500,000 cts prerequisites, and thereafter the injectiorr syringe and tlre veitl:atheter were counted (postdose counts).

Data were evaluated using a digital data analyser system (DIAG v.7.2,\íediso, Rudapest, Hungary). Twenty_min thyroid rádiophármaceutical uptake,ras calculated as the percentage of injected dose (predose counts minus post_lose counts) evaluating the thyroid regions of interesi (ROIs) in the |ast frame of:he dynamic study. ROis were drawn around itr the two thyroid lobes, salivary;lands and background region in tlre neck not containing large blood vesseIi.'I'hvroid sizes were estimated using a previously calibraled modified built-in:rogram (macro).

\lűliStics

Student'S paired l-test was used for evaluating the differences in quantita-:ite scintigraphic data ofthe two thyroid ]obes within the eutlryroid and hypo-:hl,roid groups. Student's l-test was used for evaluatiIrg tlre significance oidif-=rences between the euthyroid and hypothyroid groups. BeJause of the lorvlttrnber of cases the hypefthyroid and maIignancy gÁup. *"." not evaluated sta-:isticalIy.

Acta Iblelina.ia Hungarica ]6, I998

|50 BALoGH €t al.

Results :_-.er in this group were rh._.bes in the quantitati\ e d:

l:orvn in Table 3, uhile the

::iher 27 out of the 36 h; pr

;emples were taken in ]]:: :mpatible with endocrine:- = 2). In group (a), there r, ,

., nile in the other dogs c.'f :

_-hanges regarded as susgesi

]a the arrector pili muscles

'l]licular infundibulum and l

Ql::(n = 36, bul in i

).Ze (mean } SD, cmr)

ilrroid/Salivary gland Rol r:-..6n * SD)

-1 roid/Background ROI rat: :

'-36n + SD)_ _,-min thyoid uptake

rean + SD. l. D. %)

A total of 62 dogs with suspected thyroid abnormalities were evaluatedbetween January l995 and October l996. The age distribution of euthyroid andhypothyroid animals was similar and it shows that hypothyroidism is basically adisease of middle-aged dogs. The occurrence of hypothyroidism was four timeshigher in female/spayed female than in male/castrated dogs (29 vs. 7). The dogbreeds most commonly affected with hypothyroidism were the original Hungar-ian breed Puli (n : 6), the Schnauzer (n:5), and the Doberman, Boxer andGerman shepherd (3 each). However, the majority of dogs (l3 out of 62) weremongrels. The patients' data are shown in Table l, and typical ventral scans ofthe neck region are presented in Fig, 1.

The total thyroid size of euthyroid dogs was 3.60 + 1,36 cmj, the thy-roid/salivary gland ROI ratio was 2.01 + 0.55, the thyroid/background ROI ratiowas 3,86 + 0,90, and the 20-min thyroid radioactivity uptake was 1.17 + 0.1l%of the injected dose (I. D.) in average. There was no significant difference be-N/een the two thyroid lobes in any of the quantitative data in the euthyroidgroup, The basa| T4 concentration of healthy dogs was 33,13 + 9,64 nmol/L,which increased to 53.07 + 16.71 nmollL 4 h after TRH administration, The quan-titative data ofeuthyroid dogs are shown in Table 2, while the results ofthe TRHstimulation test in Table 6,

Table 2

Quantitative data ofeuthyroid dogs (n : 22)

Leftlhyroid lobe

Rightthyroid lobe

Total(or average)

SiZe (mean + SD, cm])ThyroidiSalivary gland RoI ratio(mean + SD)Thyroid./Background RoI ratio(mean * sD)20-min thyroid uptake(mean + SD, I. D. %)

| .69 + L14

1.97 + 0,5l

3.78 J.0.92

0.57 + 0.32

|.92 + 0,9z

2.04 + 0.47

3,94 t 0.77

0.61 + 0.2,1

3.60 + 1.36

2.0l + 0.55

3.86 + 0.90

1.17 + 0.7l

In two hyperth) roid

:he euthyroid (and also in tl

.ma]l size and cacllectic. O::l1 roid/background ROI r

r]an)- times higher in the i_

].-,rd) animals, The quantitaHistologicall;,. the r

]ro\,ed to be an adenoma

lpectively. No metastases ,

,:nlrvhere else in the bodr

lo_ss but the thyroid uptaL

nired tumour, its volutn tj8 cm3 as determined t;_i nronths after scintigrap:

tound to be 62 cm' insteat

roid malignancies are shL,\,

In 9 out ofthe 36 hypothyroid dogs absence of TcO4- uptake by the thy-roid glands was found (bilateral non-visualisation); thus, they were excludedfrom fufther quantitative evaluation. There was no significant difference be-tween euthyroid and hypothyroid dogs in thyroid size, but all otheí quantitativedata (thyroid/salivary gland, thyroid./background ROI ratios, 20-min organ up-take) were significantly lower (p < 0.00l) in hypothyroid tlian in euthyroid ani-mals. The biggest differences were demonstrated in 20-min organ uptake. Nei-

Ácfu releliharia Hungarica 46, 1998

l,HYRolD SCIN],|GRAPIlY lN DoGS

,3r in this group were there significant differences between the two thyroidles in the quantitative data. The quantitative data of hypothyroid dogs are

. ,:rrn in Table 3, while the results of the TRH stirnulation test in Table 6. Alto-_.:her, 27 o],lt of tl]e 36 hypothyroid patients showed skin abnormalities. Biopsy.lples were taken in 24 cases. They were evaluated as (a) showirrg clranges. :lpatible with endocrine disorders (tl = 22), (b) cases withottt such changes- = 2), hr group (a), tlrere were 4 cases with lesiotls suggestive of hypothyroidisrn,:lile in tlte other dogs of this group (n: l8) no such alterations were found,ranges regarded as suggestive of lrypotlryroidism were eosinophilic vacuolisation: tlre arrector pili muscles with concomitant acanthosis and hyperplasia of the

-licLrlar irrfurldibulum arrd telogenic/catagenic arrest ofthe hair groMh cycle.

Table 3

Quantitative data of hypothyroid dogs(n = 36, but in 9 cases there was bilateral non-visualisation)

Left lhyroid lobc Right th}roid lobe 'lbtál

- : {mean * SD, cm3)-_, roid/Salivary gland ROI ratio..:an + SD)-, roid/Background Rol latio- :rn t SD)

-]llin thyloid uptake-:aniSD,LD,%)

1.85 r l ,02

1.04 r 0,44

2.27 t 0.62

0,l4 + 0,17

1.90 + 0,96

1.I2 t 0.66

2.38 + 0.69

0.20 ,l 0, l3

3.15 t 1.34

1.08 + 0,56

2.32 + 0.,70

0.34 + 0.22

In two hyperthyroid dog patients smaller thyroids wele detected than in: euthyroid (and also in the hypothyroid) dogs. However, both animals were of

.'.lel] size and caclrectic, Other quantitative data, e.g. the thyroid/salivary gland,,, roid/background ROl ratios, and 20-min orgatr radioactivity uptake were]n\ times higher in the hypefthyroid dogs than in the eutlryroid (and hypothy-

::J1 anirrrals, The quantitative data of hypeftlryroid dogs are showri in Table 4.

Histologically, the two enormously large thyroid malignarrcies examined,..red to be an adenoma and a malignant epithelial-mesenchymal tumour, re-

.:ectively. No metastases were detected in the lungs (or in the mediastinum) or.,:r rr here else in the body, The ROI ratios were similar to those of hyperthyroidj ]{s but tl]e t|ryroid uptake was lower, After successful surgical removal of the..;red tumout,, its volume was measured and found to be 5 l cmj instead of:! cnl3 as determined by scintigraphy, The otlrel dog lraving adenonra died, llonths after scintigraphy. lts neoplastn was rneasured after necropsy and:'.uncl to be 62 cm3 instead of 66 cm'. The quantitative data of dogs lraving thy--:id tllaligtratrcies are slrown in Table 5.

Acla l'elerinalia Hungarica 16, l998

BALOGH et ál

Table 4

Quantitative data ofh}pefih}Toid dogs (n : 2)

Left Rightthyroid lobe thyroid lobe

Total

-_- ; study, TRH stimulation nl-::Jlsm; however, there is a th

::::ents (Rudas et aL, 199-{u: I

:trhl roidism in dogs is als,-,

-:ement or malignanc1 is nc

. :,,rld be the presence of aut,,,;

,. had no possibility to mea_t

-.;llts rvhen T4 is measured b

:J3nt concentratiorr of th; roii:rrlains why basal T1 ler els

__.e in the diagnosis of hl penh

size (mean + SD, cm3)Thyroid/Salivary gland ROI ratio(mean + SD)ThyroiüBackground ROl ratio(mean t SD)20-min thyroid uptake(mean + SD, L D. %)

0.95 + 0,05

14,00 + 2.00

16.80 + 2.40

3.15 + 0.97

0.95 + 0.15

16,00 * 2,00

18.60 + 2.20

3.18 + 0.98

1.90 + 0.20

15,00 + 2,00

17.,70 +2.40

6.33 + 0.98

Table 5

Quantitative data ofdogs having thyroid malignancies (n : 2)

Thyroid mess

Size (mean + SD, cm3)Thyroid,/Salivary gland RoI ratio (mean + SD)Thyroid/Background ROI latio (mean + SD)20-min thyroid uptake (mean + sD, I. D. %)

62 + 5.218.4 + 1 .420.2 + 1.82.11 + 1.3l

§1

Table 6

Results ofthe TRH stimulation tests (n = 62)

A)

8h4h0h

Euthyroid group (n = 22)T4 (nmol/L), mean + SD

Hypothyroid group (n : 36)T4 (nmol/L), mean + sD

53.01 + 16.11 38.13 t 12.,1433.13 x 9.64

11.96 + 5.94 14.60 + 9.4l 11.53 t,7 .42

Discussion

When determining T4 concentrations alone, a diagnostic error manifestingitself in false positive or negative results is seen in up to 50% of the cases(Rudas et al., 1994a; Rudas et a1., 1994b; Paradis et al., l995), Therefore, someform of stimulation test should be utiIised to obtain a more accurate diagnosis. In

Acla l/elerinaria Hunganca 46, ]998

c)

Fig 1 Typical ventral scilt(C) a hyperthyloid dog, ani ,I

of 99'Tc-pertechnetare in_iec

of th\Toij ,

THYROID SCINTIGRAPHY IN DOCS

lis study, TRH stimulation negativity was chosen as the criterion of hypothy-:oidism; however, there is a theoretical possibility of losing tertiary hypothyroidatients (Rudas et al,, I994ai Rudas et al., 1994ó). The clinical diagnosis of hy-

-nhylroidism in dogs is also difficult in many cases, especially if thyroid en-

lrgement or malignancy is not present. One ofthe major differential diagnosesrould be the presence of autoantibodies to Ta and T3; however, in our laboratoryae had no possibility to measure them. These autoantibodies produce spurious:e.;ults when Ta is measured by immunoassay, often resulting in an elevated ap-3aíent concentration ofthyroid hormones in affected dogs (Peterson, 1995). This:rplains why basal T+ levels and üe TRH stimulation test are not suitable forrse in the diagnosis of hyperthyroidism and thyroid malignancies.

l53

:0

8

s

l

Frg. -l, Typical ventral scintigraphic images of (A) an euthyroid, (B) a hypothyroid,(C) a hyperthyroid dog, and (D) ofa dog with thyroid tumour 20 min after 80-150 MBq

of9hTc-penechnetate injection. Note differences in the radiopharmaceutical uptakeofthyroid glands, salivary glands and background

A)

c)

Acla l/elelinaria Hungarrca 46, 1998

BALoGH et al

. :O1 scintigraph} can p:

. _ i canine thyroid abnorn-,E, _ morphology (contours. .:.:::: surgery or chenrothera;- i 3nd T,B ROI ratios and ri

This work was suppc::._ _ L\). b.v grant no j9-] .

-_:

-::t (OMFB), and b1 PhD _,

,,rB) The authors thank \í:.- . : in the practical works arrd :

-:.ms, W. LI , Daniel. C B :

pertechnetate th},roid ja:::

]:.i. K A,, Hornof, w J :j.: _-

peítechnetate imagin! :i:10 norma] cats Vet R3a:a

-]:-lsha\v. B, E, and Rijnberk. \of primary hypoth) roii: :.

,a:rard. P and Regnier. A (l9sq193 l98,

].r\ ner, W. R, (1996): Thl r.,:J .:clear Medicine Noíh C3.-

_-i3stain, C, B and Pancierl ]and Feldman. E C le:.ders co, PhlladeLphie ::

:,, inger, J, V,, Nelson. R \\ j-- ]tion testing in health) c._i

:.rrrest, L, J, Baty, C. J. \íer.3]:,of treatment using \ o ,_::

14l 145

irank. L, A (l996): Conrpe::.i_tion for evaluating th1:l :

Gloss, T, L,, Ihrke, P ] and \\.17) ln: Gross, T L. ;..:

Mosby Year-Book S: -_Grunau, B., Nolto, I. and Hop:..

Hundes, Kleintierpra\:s,+Hammerling, R., Leidinder. K -

agnostik unter PrL\i b.]21,1-356

The results of the histopathological examinations of skin biopsies seemedto be helpful in terms of signalling possible endocrine disorders, This rnethodwas included in this paper to underline its potential usefulness in the diagnosisof hypothyroidism.

Thyroid volumetric analysis has been elaborated by Forrest et al. (1996)for radioiodine dose calculation in cats, but no similar data are available fordogs. In nine out of 36 cases we could not estimate the thyroid size because ofthe absence of radiopharmaceutical uptake by the thyroid glands, and the re_mairring 25 hypothyroid animals did not differ significantly from the euthyroidones. This suggests that thyroid hypofunction is independent of tlre volume ofthe tlryroid glands. It was surprising also for us that, in contrast to hypefihyroidcats where thyroid volume is generally many times larger than in euthyroid cats(Wisner et al., 1994; Forrest et al,, 1996; Peterson, 1995), in two of our hyper-thyroid dog patients the total thyroid volume was smaller than the average thy-roid volume of euthyroid and hypothyroid dogs. This might be due to the factthat our hypefthyroid dogs were extremely cachectic but both showed the char-acteristic clinical signs (weight loss, increased appetite, heat intolerance, rest_lessness, tachycardia) of hyperthyroidism,

It is a fact that static thyroid scanning is more easy to perform and doesnot always require the sedation of animals. The advantages of dynamic thyroidscintigraphy have been reported only in cats but not in dogs. Beck et al. (1985)estimated thyroid/salivary gland (T/S) and thyroid/background (T/B) ratios inhealthy cats of different age. The T/S ratio was found to be nearly 1 and the T/Bratio was between 2 and 3, In contrast, in this study approximately twice higherT/S ratios and an average TlB raíio of 3.86 were found in euthyroid dogs. Thisstudy, however, differed from that of Beck et al. (l985) not only in the speciesstudied but also in the time of starting the examination after 99.TcOo- injection(10-15 min vs. 20 min) and in the position ofbackground ROIs (shoulder versusneck). Although technetium pefiechnetate is an isotope less frequently used forevaluating radioactivity uptake of the thyroid glands tlran l3II-iodine, Nap et al.(1994) published the one-hour TcOa uptake in euthyroid (range: 0.8-3.9 %, me-dian : 2.2%) and hyperthyroid (range 5.2)3.9 %o, median = 14.S%) cats, andtheir data show good agTeement with our results. In euthyroid dogs we obtainedapproximately the same quantitative data as Adams et al. (1997) in their mostrecent work,

In summary, we believe that simple static scintigraphy a|one can lielp inmany cases when a hypothyroid dog shows absence of thyroid radioactivity up-take or neoplastic enlargement. However, thyroid abnormalities are much moredifficult to diagnose when the clinical symptoms are not sufficiently relevantand the laboratory findings are somewhere at the border]ine of non-physiologicalva|ues. our results suggest that thyroid volumetric measurement and functional

Acla yelerinaria Hungaica 46, ]998

I55

TcOa scintigraplry can provide valuable additiorral information in the diagno-. . ol canine thyroid abnormalities, and it is especially suitable for assessing thy-- :J morphology (contours, size, location, possible ectopic Iobes or metastases)]tr-.lrre surgery or chemotherapy and for evaluating tlryroid function on the basis of- S and T/B ROI ratios and the 20-min radioactivity uptake ofthe thyroid glands,

Acknowledgements

This work was supported by grant no, F-o17462 of the National Research Fund, iKA), by gfarú no. 49-7174 of the National Committee for Technological Develop-

-:nt (OMFB), and by PhD research grants of the University of Veterinary Science\ KB), The authon thank Mrs L, Dimer and Ms Zsuzsama Suhajda for their valuable3lp in the practical Works and for careful preparation ofthis manuscript,

References

'.]ams, W H,, Dani9l, G, B., Potcrsgn, M G, and Young, K (l997): Quantltative 99''Tc-

peíechnctate thyroid scintlgraphy in normal Bcagles, Vet. Radiol. 38, 323-328,].ck. K.

^,I]ornot W, J. and Feldman, E C (l985): Thc normal feline thyroid Technelium

pertcchnetate imaging and determination of thyroid to sa]Ivary gland mdioactiVily ratios inl0 normal cats Vet, Radiol,26,35 38,

3e]lshaw, B. E, and Rijnberk, A, (l979): Radioimmunoassay ofplasma To and T3 in the diagnosisofprimary hypothyroidism in dogs J Am Anim Hosp. Assoc, 15, 17 23,

3enard, P. and Regnier, A (1989)| Scintigraphie thyroidienne chez le chien, Rev, Med. Vet 140.l93-198

3ra\vner, W R. (l996): Thyroid and parathyroid imaging. In: Berry's Handbook of Vetcíinary Nu-clear Mcdicine, North carolina University, Raleigh, North Carolin a pp .7l '19

Chastain, c. B. and Panciera, D L (l995): Hypolhyroid diseases (Chapter ll5) In: EttiDger, S Jand Feldman, E, C (eds) Texlbook of veterinary lnternal Medicine, Vol. II W. B Saun,ders Co, Philadelphia pp, l487 150l,

E\inger, J, V., NeIson, R w and Bottoms, G, D. (l985): Thyrotropin-releasing hormonc stimuia-tion testing in healthy dogs Am. J vet Res,46, 1323-1325.

Forrest, L, J,, Baty, C, J, Metcaif, M, R, and Thrall, D, E, (l99ó): Feline hyperthyroidism: efficacyof treatment using volumetric analysis for radioiodine dose calculation, vet Radiol 37.14l l45,

Frank, L A, (l996); Comparison of thyrotropin_releasing hormone (TRH) to thyrotropin stimula-tion for evaluating thyroid function in dogs, J. Am, Anim l,Iosp Assoc 32,481-487.

Gross, T L., lhrke. P, J. and Walder, E. J. (1992). ^trophic

diseascs of the hair íolliclc (Chapter17) ln: Gross, T L,, Ihrke, P J. and Walcler, E. J, (eds) Velerinary DermatopaihológyMosby Year-Book, st, Louis, pp,274 275

Grunau, B, Nolte, L and Hoppen, H-o. (l995): Der TRH-test zur Diagnose der Hypothyreose desHundes. Kleintierpraxis 40, 515-52l,

Hammerling, R, Leidinder, K and Failing, K. (l992): Vergleichende studien zur Schildddsendi_agnostik unter Praxisbedingungen

- T4, fT4, TSH-test und K-werte. KleintierpÉxis 37,2,7,7-356

THYRoID SCINTIGRA],HY IN DoGs

Led

odsis

6)brof

id)fdts

l,-

Acla leleriharia Hungdr]ca :.. l rr;

l5ó BALoGH et al

Ilightowor, D. (l986): Veterinary Nucl§ar Medicine. semin, Vet. Med. Surg, (SmaII Anim) l,108_120,

Jcffers, J, G. (1990): Recognizing and managing thc effects of canine hypothyroidism vet, Med,1,2,

1294_1308 .

Kinlzer, P, P and Peterson, M, E, (l99l): Thyroid scintigraphy in small animals. Semin. Vet-Med, Surg, (Small Anim.) 6, l31-139

Lothrop, C D , Tamas. P M and Fadok, V. A. (1984): Canine and leline thyroid function asscss,ment with thc thyro1lopin-rcleasing hormone response test, Am, J, Vet, Res 45, 23l0-23|3

Marks, S, L. Koblik, P, D., Hornof, W. J. and Feldman, E. c, (l994): g"Tc-pertechnetate

imagingofthyroid tumors in dogsi 29 cases (l980-1992), J Am. Vet, Med, Assoc,204, ?56-?67,

Nap, A, M. P,, Pollak, Y, W, E, A, Brom, W, E, and Rijnberk, A (1994): Quantitative aspecls ofthyíoid scintigraphy with pertechnetate (9'Tco4J in cats, J. Vet, Int, Med, 8,302 303.

ollver, J W, and Held, J. P. (1985): Thyrotropin stimulation test - new perspective on value ofmonitoring triiodothyronine. J, Am Vet Med Assoc 187,93l,934.

Paradis. M.. l-epine, S,, Lemay, S, and Fonlaine, M, ( ]991): studies ofvarious diagnostic methodsfoí canine hypothyroidism. Vct. Dermato1.2, l25-132

Paradis, M., Page, N,, Lariviere, N. and Fontaine, M, (1995): serum íree thyroxine concentrations,measured by chemiluminescence assay before and after thyrotropin administration inhealthy dogs, hypothyroid dogs, and euthyroid dogs with dermatopalhies. can Vct. J, 37,289-294.

Petcrson, M. E, (l995)| Hyperthyroid diseases (chapter l 14) In: Ettinger, S. J. and Feldman, E. c.(eds) Textbook ofVeterinary Internal Medicine Vol, II W. B, Saundels Co., PhiladcIphia.pp l466-1487,

Rijnberk, A (l996): Thyroids, Protocols for function test§, In: Riinberk, A, (ed.) clinical Endocri-nology of Dogs and cats - An Illustrated Text, Klurver Academic Publishers, Dordrecht.nn ]§_iq ?n5_, ] ?

Rudas, P., Barlha, T,, Tóth, J, and Frcnyó, V, L ( l994a)| Impaired local deiodination of thyroxjncto triiodothyronine in dogs lvith symmetrical truncal alopecia Vct. Res, comm, lE, l75l82.

Rl]das, P,, Papp, L, Hu§Zenicza, Gy., Bartha, T and Berkényi, T (1994b)| Reflections on thejudgement of symmetric a|opecia of endocrine origin in dogs (in Hungarian, wilh Englishabstract). Magyar Allatoru osok Lapja 49,'120-'729

Scolt, D. W,, Miller, W, H, and Criffin, C, E. (l995)l Endocrine and metabolic diseases (Chapter9), In| scott, D, W,, MiIIef, W, H, and Griífin, C, E (eds) Small Arrimal DerInatology 5'h

ed W. B. Saunders Co, Philadelphia, pp.628-119.Sjollema, B, E,, Pollak, Y, W D. A,, Brom, W. E. and Rijnberk, A (l989): Thyroidal radioiodinc

uptakc in hyperthyroid cats Vet, Quart, 11, l65 l70Wisner, E, R,, Theo, A. P,, NyIand, T, G and l{ornof, W J (l994): Ultrasonographic examination

of thc thyroid gland ofhypcrthyroid cats: Comparison to oo''I'coo- scintigraphy Vet, Ra-diol.35,53-58.

,4c!a l/elerinaria Hungarica 16, I998

DLIG\OSIS oF H\ PIlJ\tP,{IED To HL\t-\

JuIianna THLRa"-,],, . _

íReceir cJ O:: :

Spontaneous a,1-.crine disorder occun.;: ::

rests on the documelt.::::or blood The diagni: , .

iionr lruman methods :::; :

In some cases the di,r-:i]:rhe modtfication oi :::-,:phatase (AP) and ils :e::-ogenous glucoconicojl .1

values in moming urir-3 .ences in urine conca:,::::

(DX) at a relati\ el) .",, _

Secretion of ACTH r. ;as 24 to 48 h. Tlrere-cl:cal tumour would n.,: :=its administration Th: :,

on the observation íh:: ,_.

ruitary ACTH and thi\ .of its dose, dexameth3s ]:combined With the lne--ing urine samples on ::-.ratio one has to s;..-suppressible pituit3n - J::

mour arising f,otn th:

quently used lecllniqL: :

ever, it has the Sa]ne 3,.

cortical diseases as in i.

Key words: H T

phatase (ALP), stero.c-

adrenocorticotIopic h.--.

1,

ed

ng

of

ls,ln1,

l-Lt

le

/et,

of

5s-

0

e

h

Ir

Acla yeíeri aíia Ilunga|ica 46 (2), pp 157 l73 (l998)

DIAGNOSIS OF HYPERÁDRENOCORTICISM IN DOGS ASCOMPAR.ED TO HUMAN DIAGNOSTIC METHODS: A REVIEW

Julianna THuRöcZy', L, B,e.LoGH', Gy, HUszEN]cZAl, Gy, A, JÁNoKI2

and Margit KuLcsÁnr

'Department ofobstetrics and Reproduction, University ofveterinary Science,

H-l ioo Budapest, P.o. Box 2, Hungary; 2Department of Applied Radioisotopes,\ational 'Frédér,ic Jo]iot-Culie' Research lnstitute for Radiobiology and Radiohygiene,

H-1775 Budapest, P O, Box 101,I1ungary

(Rcceived October 3, 199?; accepted January 28, 1998)

Spontaneous hyperadlenocorticism is a common and well-recognised endo-

crine disorder occurring in dogs, The biochemical diagnosis of hyperadrenocofticism

rests on the documentation of excessive glucocorticoid levels or metabolites in urine

or blood. The diagnosis of canine hyperadrenocofiicism is historically developed

from human methods and based on non-dynarnic and dynamic tests and visualisation

ln sonre cases the different ranges ofendocrine palameters or nretabolites necessitate

the modification of human protocols. Elevation of the activity of alkaline phos-

phatase (AP) and its heat-Iesistant isoen4rme (SIAP) induced by endogenous oí ex-

ogenous glucocorticoid excess raise the suspicion of hlperadrenocorticism, Cortisol

values in morning urine are related to cleatinine concentlations to correct for differ-

ences in urine concentration, Theoíetically, the admjnistration of dexamethasone

(DX) at a íelatively low dose (0.01 mg/kg of body weight) can inhibit the pjtuitary

secletion of ACTH and, in tum, decrease endogenous coíiso] secretion for as long

as 24 to 48 h Thereíbre, Dx administration to dogs with a functioning adrenocotli_

cal tumour would not affect the plasma cortisol concentration at any tine following

its administration. The high-dose dexamethasone Suppression test (HDDS) is based

on the observation that the function of adrenocortical tumours is independent of pi-

tuitary ACTH and üey completely suppress ACTH secretion; therefore, regardless

ofits dose, dexamethasone is never able to suppless cortisol secretion. HDDS can be

combined with the measulement of urinary cortisol/creatinine (c/c) ratio fiom mom-

ing urine samples on thlee consecutive days, In case of non-suppressible ut,inat1 c/c

Iatio one has to speculate on differer-rtiating adrenal tumour (AT) from non-

suppressible pituitary-dependent lryperadrenocorticism (PDH) due to a pituitary tu-

mour arising from the intermediate lobe. Radiocholesterol scintigraphy is a less f,,e-

quently used techniqrre in the diagnosis of canine Cushing's syndrorne (CCS); how-

ever, it has the same advantages in the ]ocalisation and characterisation of adreno-

cortical diseases as in humans,

Key words: Hyperadrenocorticisnr, diagnosis, humans, dogs, alkaline phos-

phatase (ALP), stefoid-induced ALP (SIAP), urjne corlisoVcreatinine (c/c) ratio,

adrenocorticotropic hormone (ACTH), dexamethasone suppresSion, scintigraphy

0236 6290/98/S 5 00 o )998 Ákadémiai Kiadó, RuLldPesl

THURoCZY et aI D.:.a., _ ! !

The conisol creatin i:,É, : ,,. sensitire tes1. CL.n]s:._]_:. Ihis ralue does n..t r a:-,

:-:: Contteras et al . 19S6--.:: ;ab]e because ol the ]:i_-:::]nlne,

ln addition to the m:3j _

- :3:est to determine me:a'r:- , :::ricortisol, tetrah\ dI.::

-_,:h reflect about 50 per c_l

: :::ten et al., 1969: \'ac,.-:a ::Plasma steroids t t:,r::

.,:i as dehydroepiandrt s:.l:l \S) and androstened.,::.:

. _:::id secretion of the ]:u:-l::

':: the synthesis of teste.si=::

: _ I991b). The averaee pr::-:rrel1 by sexual maturatic:,:::trse. one-third to one-f-- u:_

: _sile adrenal origin ani i::- : r diurnal variation (Ro::r i:

- ]ji important androgen s::::: DHAS, its level shorrs J,_f.e.senfeld et al., 197l ),

Recent studies har e r;: -.nisol is influenced br er.,

::ease in plasma cortisoI :l.:..rmal subjects and patien:s

:rree times higher than nor,:l

:i .1981; Lacroix et al,. ]99j--.]niso] values ir-r Cushinc's.:he stress-Ie]ated itlcrease j:.L.llection from 4S-hour hc sc_ 1:00 p.m. value or less thaisampling from 1:00 P,]tí, til] -

A well-establishe d metdetermination of plasma ACT,rCTH in a patient rrith nr-,r-

adrenal lesion (adenoma o:;be caused by an ACTH-p:.-:rion. Its distinction is nor re,:;

Spontaneous hyperadrenocorticism is a common and weIl-recognised en-docrine disorder occurring in dogs (Rijnberk et al., 1968ó). The underlyingcause of canine hyperadrenocorticism is most frequently adrenal hyperpiasiásecondary to an ACTH-secreting pituitary tumour or less frequently , p.i-u.yadrenocortical tumour, Tlre reported incidence of pituitary tumours in dogs is85-90 per cent, Although adenomas of anterior lobe origin are most common(75%), tumours of the pars intermedia may also be associated with canine hy-peradrenocotticism (Rij nberk, l 996).

The clinical features of hyperadrenocorticism are caused by abnormallyhigh circulating concentrations of glucocorticoids. This glucocorticoid excessmay also be due to exogenous administration, so lryperadrenocorticism is oftenialr9cenIc, Suspicion of spontaneous hyperadrenocorticism arises on recognitionofthe clinical signs, and its diagnosis is established on the basis of abnorrna|itiesin adrenal function tests. Basically the reason for these abnormalities is eitherdecreased sensitivity of the hypothalamic-pituitary axis to the negative feed-back effects of cofiisol excletion or AcTH-dependent excessive cofiisol releasefrom the adrenal cortex (Rijnberk, l996).

The physiology of secretion and the metabolism of different adrenal ster-oids are similar in dogs and humans. On this basis, the dynamic adrenal cortextests used in dogs are modified human test methods. The aim of the present re-view is to summarise the human diagnostic methods and their usefulness irr theveterinary practice, supported by data from our own laboratory and by those fromthe literature.

Diagnosis of hyperadrenocorticism in human subjects

The biochemical diagnosis of hyperadrenocoIticism rests on the docu-mentation of excessive levels of glucocorticoids and/or their metabolites in urineor blood. No other tests are specific and therefore useful in diagnosis.

A. Non-dynamic tests

Urinary cortisol and cortisol metabolltes, Radioimmunoassay (RIA) deter-mination of urine free cortisol (UFC) in a 24-hour collection of urine is probablythe best and simplest non-dynarnic screening test for hyperadrenocorticism. Thedetermination of unbound urine cortisol concentration is not affected by othermedical conditions, The basic condition of tlre measurement of uFc va|ue is theentire collected 24-hour urine output, therefore under- or overcollection of urineyields spuriously low or high results. Compliance with urinary collections causesproblems, and many attempts to improve them have been made to eliminate errorsof sample collection, including disturbed sleep period ofpatients (Crapo, 1979).

Acta l/elerinalia Hungarica 16, 1998

DIAGNOSIS OF HYPERADRENOCORTICISM lN DOGS

The cortisol/creatinine (c/c) ratio determined in early rrrorning urine is a_- sensitive test. Cortisol excretion is corrected by creatinine excretion, be-, ,. :his value does not vary more than 1 5 per cent from day to day (Walker,,-, aontreras et al., 1986), Determination of c/c ratio in 24-hour ttrine is not,_, :able because of the lack of paral|elism in the excretion of cortisol and, ,-lne,

ln addition to the measurement ofUFC, there are tests largely ofscientific:-j:t to determine metabolites of cortisol such as 20-dihydrocortisol, 6-,- ricortisol, tetrahydrocortisol arrd l7-hydroxicorticosteroids (l7-0HCS), _-. reflect about 50 per cent of cortisol production (Porter and Silber, l950;,:j:.n et al., l969; Vaccia et al., 1979; Phillipon, 1982).

Plasma steroids (cortisol and adrenal androgelrs). Adreria| androgens., as dehydroepiandrosterone (DFIA), dehydroepiandrosterone sulplrate:: \S) and androsterredione (Aa-A) corrrprise a considerable fractiorr of the.- :d secretion ofthe human adrenal glands. Tlrese compounds are preclll-sors, ::,c synthesis of testosterone and oestrone (Hanirrg et al., 1991a; Hallirrg et

q9]ö). The average production ofDHA and DHAS is influenced by age,^-:l. by sexual matulation, and its secretion slrows a marked age-related de-..se. one-third to one-fourth in the seventh decade. DHAS has an almost ex-_. i e adrenal origin and its plasma level is relatively stable, showing only mi-- JiLrrnal variation (Roserrfeld et al., 1915). DIIA is quantitatively the second.: illlportant androgen secreted by the adrenal coftex and, in contrast to that]HAS, its ]evel slrows diurnal varjation largely parallel to coftisol secretion

: s.,nfe]d et a|., l971).Recent studies have revealed tlrat the norma| diurnal rlrythm of plasma

::_-ol is influenced by environmental effects. Meal-related, stress-related irr-

.:se in plasma cortisol may result in apparently elevated concentrations in,:ral sLrbjects and patients witlr psychosis may have cortisol values one to

,:j times higher than normal (Sacher, 1975; FolIenius et al., 1982; Halnet et- 987; Lacroix et a|., 1993), Moreover, tlre normal diurnal variation of plasma

- :sol values in Cushing's syndrome is blunted or evel absent, Elirrrinalion of. Stless-related increase in plasma cortisol concentratioll is difficult. Sample

- ..tion from 48-hour hospitalised patients disturbs the sleep using criteria of,]0 p,m. va|ue or less than 8:00 a.rn. value. An alternative method is multiple

.:lpling from l:00 P.M. till 4:00 P.M, (Desai et a1., 1987),A well-establislred method of testing pituitary-adrenal axis function is the

:::ermination of plasma ACTH Ievel. Persisterrtly undetectable levels of plasma, aTH in a patient with non-iatrogenic )ryperadrenocorticisrn indicate a prinrary:renal lesion (adenoma or carcinoma). An elevated plasma ACTH value may

-. caused by an ACTH-producing pituitary tumoul or ectopic ACTH produc-]:r, Its distinction is not re|iable, therefore dynamic tests are still necessary,

Acla l/elerinaria Hungarica 16, l998

l60 THURÓCZY et al

B. Dynamic íests

Dexamethasone (DX) suppression lasls, DX is a glucocorticoid receptoragonist, which does not cross-react with cortisol iri RIA, therefore it is very use-ful for estimatirrg whetlrer the normal mechanisms of negative feedback are op-erative. In some human cases the reasons for abnormal cortisol production orlack of DX suppressivity are associated with tlre use of oral contraceptives con-taining oestrogen, endogenous depression, or alcoholism (Butler and Bessr,1968; Rees et al,, l977; Nickelsen et al., l989).

Low-dose dexamethasone suppression test (LDDS) is the best known oftlre dynamic screening tests for suspected lryperadretrocorticism. ln the first in-terpretatioI,I of this test l7-0HCS excretion is tneasured in 24-1rour collectedtlrine during admiriistration of 0.5 mg DX every 6 hours for 48 hours and theurine collection is repeated on the second day. l7-0HCS excfetion higlier thanl l prmol/day is considered to indicate hyperadrerrocorticism. As there are diffi-culties with compliance irr urine collection, the overnight LDDS is preferable(Liddle, l960). Plasma cortisol is measured at 8:00 a.m. after an oral dose of1 rng DX was given at l 1:00 p.m, or l2:00 midnight tlre night before, No base-Iine measurement is required, and if suppression of plasma cortisol is l80 nmol/Lor |ess, it is considered normal, while patients with hyperadrenocorticisnr give adifferent response.

Tlre best-establislred dynamic test to differentiate adrenal from pituitarylesions is tlre lrigh-dose dexamethasone suppression test (HDDS) (Liddle, l960).After a baseline 24Jrour urine collection, the patient takes DX, 2mg every6 hours, and repeats the collection of urine on tl]e second day. Suppression ofl7-0HCS to 50 pef cent or less of baseline excludes adrenal adenoma and carci-noma. False-positivity occurs in up to 6 per cent of the patients with ectopicACTH and 25 per cent of those with nodular adrenal hyperplasia (Crapo, 1979).An alternative that is suitable for differentiation is the overnight HDDS, inwlrich plasma cortisol is measured at 8:00 a,m., the patient is given 8 mg of DXat 1l:00 p.m., and another plasnra coftisol determination is done at 8:00 a.m. thenext day. Suppression of plasna cortisol to 50 per cent or less of baseline indi-cates a pituitary lesion (Bruno et aI., 1985).

Metyrapone leJí Metyrapone blocks 1 l -hydroxylation of deoxycorlisol,resulting in an accumulation of 1 l -deoxycortisol, and plasrna and urine concen-trations of cortisol metabolites. Although rnultiple studies lrave confirmed thatthe metyrapone stimulation test well differentiates the pituitary and adrenal ori-gin of lryperadrenocorticism, it produces side effects and the discrinrination ismore easily made by determination of basal plasma ACTH level (Sindler et al,,1983; Malchoff et a|., l988).

- ,t ticott,optn,re,. -l. -,

. CRH (e-,CRH t :t::*-,

_ _,: :enl h} peIadre n!,c !, r-:

-.. letain responsir ere.s_ _.::_,:s and callnot resP!-: ,

_ '_,:nt to inhibit the n:t- ].::,lous ilrjection cf cC!

._- n interyals orer ] h, \- ]nareases in plasrlla A,

_ "=:j..n

do not (Níalcho:i .-: ::je because of greater ;_ : trnd has recentl) been i

-,: prepalatiolr (Farta e: :

. ,: and 30 min) ol at -::

..i: ]0 per cent increase ::,

, ::.ates Cushing's disers:. -::se of at least 50 per cert

_, j Crapo, l990).Bilateral htJcrir,,t i. :

- _,:edure are differentia:i:- ]:i]ic tests are contradiaIa:,. jiology is norrnal but ,j)-

J,] l ),The test exploits t:::

:.rnd into the correspond :

parately via a fenloral .,:

iued simultaneoLtslr ir.,r::

: and l0 min after the in::

:nd Doppman, l99l ). ACTC uslring's disease and in;:

:retion by the corticotr!,pl_

.rease after CRH, A rati(, :

Tn contrast, pre-CRH grao

.ase and ectopic ACTH :

.\CTH secretion rrere bel.

e ase (Oldfield et al.. l99ability to localise the turli

,4cl0 lblerinaria Hungarico 16, l998

ptor]se-op-lor]on,

ssr,

rofilr-tedthe1an

ffi-bleof

Se-yL3a

)i,n-

rry0).)ryofci-lic)in)Xheti-

at-i-

isl,,

DlAGNOS]S oF lIYPERADRENOCORTICISM ]N DoGs

Corticotropin-releasing hormone (CkH) stimulation /esl. The use ofre CRH (oCRH) stimulation for the differeritial diagnosis of ACTH-

:.:endetrt hyperadrenocotlicism relies on the assumptions (1) that cofiicotlopi-. rlas retain responsiveness to CRH, while noncofticotroph tumours lack CRH

,:-eptols and cannot respond to the agent, and (2) that hypercorlisolisrn has been-_-tlcient to inhibit the nornral corticotropic response, After 1 pg/kg or l00 pg,::avenous injection of oCRH the plasma is sarr-rpled for ACTH and cortisol at

: -min intervals over 2 h. Most of tlie patients with Cushing's disease respond

.th itlcreases in plasma ACTH or corlisol, while patients with ectopic ACTH;.;retion do not (Malclroff et al., 1988). oCRH has beerr preferred to the lruman

::ptide because of greater potency, but human CRH is now more readily avail-.:le and has recently been shown to offer equal serrsitivity and specificity to the

,, ine preparation (Faria et al., 1990). Increase irr meari post-CRH ACTH values:i 15 and 30 min) of at least 34 per cent indicates Cushing's disease, and an at

=est 20 per cent increase in the rrrean posrCRH cortisol value (at 30 and 45 min)rJicates Cuslring's disease (Nieman et al., 1993), Other studies repoíed atl in-:rease of at least 50 per cent in ACTH or at least 20 per cent in cortisol level (Kaye:nd Crapo, 1990),

Bilateral Inferior Petrosal Sinus Sampling (1P,§,§/. The indications for the

:rocedure are differentiating ectopic from pituitary sources of ACTH wlreri dy-

]anlic teSts are contradictory, and lateralising a pituitary adenoma when pituitary:adiology is normal but dynamic tests indicate a pituitafy source (Oldfield et al.,

. 991).The test exploits the nonnal venous drainage of each half of the pituitary

gland into the corresponding petrosal sinus. Each petrosal sinus is catheterised

separately via a femoral approach, and b|ood for measurement of ACTH is ob-

rained simultaneously from each sinus and a peripheral vein twice before and 3,

_; and 10 min after the intravenous administration of 1 pglkg of oCRH (Millerand Doppman, 1991). ACTH concentrations are gleater in the central samples inCushing's disease and increase after CRH admiriistration, reflecting ACTH se-

cretion by the corticotroph adenoma, ACTH values in the central vein do not in-crease after CRH. A ratio of the central to peripheral ACTH values is calculated.In contrast, pre-CRH gradients have been repofted similar in both Cushing's dis-

ease and ectopic ACTH secretion, the maxima] post-CRH gradients in ectopicACTH secretiorr were below the respective minimal gradierrts in Cushing's dis-ease (Oldfield et al., 1991). Ari additional advantage of IPSS is its potential

ability to localise tlre tumour to the right or left side ofthe pituitary gland,

Ácía lbrcrlnara HunFarlca :r . ,;:

l62 THUROCZY et al.

C. Visualisation and localisation oí the tumour

Ultrasonography, Computed Tomography (CT) and Magnetic ResonanceImaging (MRI). Although adrenal glands can be visualised by ultrasonographictechniques, scintigraphy, CT and MRI have largely replaced ultrasonography inthe evaluation of functional adrena] disease.

The advent of CT scanning of the abdomen has allowed highly sensitivedetection of adrenal abnorrnalities. However, performing a scan before the adre-nal is clearly identified as the pituitary cause ofthe Cushing's syndrotne may bemisleading, The high incidence of incidentalomas ofthe adrenal at post moltemhas been confirnred using CT (Osella et al., 1994). Very few ofthese have func-tional significance.

CT scanning has not influenced the investigation for Cushing's disease inthe way that it has revoluiionised other neurosurgical problems, Its low sensitiv-ity of 47% for ACTH-producing pituitary tumours is attributable to their smallsize atld tlre failure of many of these tumours to enhance with contrast material(Kaye and Crapo, 1990; Reincke et al., 1992). That aside, a positive CT scan isstill very helpful to the surgeon, particularly if MRI is not locally available.

MRI scanning perfonns muclr better than CT in the investigation ofCushing's disease. The overall sensitivity is 72oÁ without gadolinium enhance-mel]t (NeMon et al,, 1989). The sensitivity can be improved using gadoliniumcontIast agent.

Scintigraphy. Radiocholesterol scanning performed either with r]lI-

Iabelled or "Se-labelled clrolesterol derivatives can be a very sensitive and spe-cific method irr the visualisation of tlre adrenal cortex. The technique invo|vesthe advantages of so-called physiological imaging, because after intravenousadrninistration of both radiopharmaceuticals they incoryorate into the intraglan-dular biosynthesis of cholesterol. This occurs over a period of several days sothai repeated scintigraplric examinations must be obtained from days 1 to 7 afterradioplrarmaceutical administration. Normal glands are usually not imaged, InCushing's disease bilateral uptake suggests an ACTH-deperrdent nodular hyper-plasia of both glands or, less commonly, an ACTH-independent disease (e.g.primary pigmented nodular adrenal disease, massive macronodular hyperplasia).Unilatetal visualisation of the adrenal cortex indicates most frequently autono-mous coÉisol-producing adenomas with contralateral suppression. Adrenal car-cinoma shows bilateral |ow uptake, because the greater size of these tumours(relatively undifferentiated, nonfunctiona| characteristics) does not mean ahiglrer production of corticosteroids (Hay et al,, 1996).

Acla l|ele ]lalia Hunqalica 16, 1998

DiAc\ - ]

Diagnosis o1

llthough diagrrosis of ;

: ] ]irrffi human meThLrds, .

_,:::rs oI metabolites in s.lr:

- ": Jllitlg íests

1lkűlúE phosphotilr. :-

. , :.re hl drolysis of phospi::,-. ]'loratory findings is an ;:,. _.:J AP activity in 55 per ;:

He at-resis íant -4P iri : :"

] - - _iuS glucocorticoid excess

l rlple lnethod for disting,_

_-].:3r stabjlity of SIAP ar l_ .:. the finding of ]lrcre3iii: : during anticon\ uls3]:

' - l), In our patiet-tts rrit:_

. : AP is specificall1 the st.Urinary coríicosrerú:.i

:. :Jrenocorticism in the p;.::::on to provide an integr.::

_ -: icosteroids. TraditionaL],.

',ring a diagnosis, Altht],-S:-3;..gnised as inherent \\ ith i..iing ufine fol this test has ]

Dogs with hyperadre:.,r of 17-0HCS excreted in]ó8á; Siege], 1968), AddiiIloímal dogs was shoun i

lur urinary 17-0HCS erc:]67; Rijnberk et al,. 1968.r

Measurement of LFC:].-'n and chromatograph} T']utjne purposes. Cortise.l ie

:Ssay can easiIy be pertbrm::ations suffer from interierer_.i increased plasma concer,i:

.ortisol may be expected. ,j _

jnextracted urine fronl ncr

e

c

l

DlAGNosIs oF 1-1YPERADRENoCoRTICIsM IN DoGs

Diagnosis of canine hyperadrenocorticism

Although diagnosis of canine lryperadrenocoticism is historically devel-::ed from human methods, because of the different ranges of endocrine pa--:meters or metabolites in some cases, the human protocols have to be modified.

: screening tesls

Alkaline phosphatase (AP) acliyily. The group of serum AP enryInes cataly-.:s the hydro|ysis of phosphate esters, and the most common abnormality in rou-- re laboratory findings is an increase in serum AP activity, We have found an in-_.eased AP activity ín 55 per cent ofdogs with hyperadrenocorticism (Fig, l).

Heat-resistanl AP aclivity. lt is only in the dog that the endogenous or ex-::enous glucocofticoid excess induces a heat-resistant isoenzyme of AP (SIAP).r. siniple method for distinguishing between the two isoenzymes of Ap uses the_,eater stability of SIAP at 65 "C. The test is considered quite sensitive, how-.,.er. the finding of itrcreases in SIAP is nonspecific, SIAP may be elevated in:lgs during anticonvulsant therapy, in diabetes mellitus and hepatopathies;ig, l), hr our patients with hyperadrenocorticism 66 (+ SD 24.2) peí cent of

::eir AP is specifically the steroid-irrduced fraction (Fig. 1) (Teske et al., 1989).Urinary corlicosteroids. The cornerstone in the diagnosis of hurnan hy-

:: radrenocorticism in the past decades has been the 24-hour urine sample col-:ction to provide an integrated assessment of the amount of produced urinary

_ _.nicosteroids. Traditionally it has proved to be the most reliable means of con-:.rnring a diagnosis. Although the test is informative, and despite the advantages::cognised as inl]erent with this diagnostic tool, the cumbersome nature of col-3ctil]g urine for tlris test has rnade it rarely used in dogs.

Dogs with hyperadrenocorticism have a significant increase in the quan-::q of 17-0HCS excreted in their urine over a 24-hour period (Rijnberk et al.,9ó8á; Siegel, 1968). Additionally, administration of exogenous ACTH or DX

:_. normal dogs was shown to result in a respective increase or decrease in 24-:.lur urinary l7-0HCS excretion compared to basal excretion (Wilson et al.,967; Rijnberk et al., l968a).

Measurement of UFC includes several purification steps, such as extrac-:iLln and chromatography. Therefore, these determinations are not suitabIe for:.rutine purposes. cortisol deterrninations in unextracted urine by radioimmuno-]ssay can easily be performed as a routine procedure. However, these determi-rations suffer from interference by other steroids (Murphy et al., l981), In cases-.f irrcreased p|asma concentratiorrs of cortisol a disproportionate rise in urinary:ortisol may be expected, due to an increased percentage of unbound cortisol. In]nextracted urine from normal human subjects the values of immunoreactive

Ácla Iblelinalia Hunqarica 46, l998

l64 THURoCZY et al

cortisol are about six times higher than those obtained after extraction. Most ofthe interfering compounds have a polarity similar to that of cortisol, wlrichmakes it impossible to eliminate them by simple solvent extraction procedures

However, removal of these compounds may not be essential for diagnostic pur-

poses, since a considerable part of them may represent metabolites of cortisol, ]n

addition, even after chromatogíaphy it is uncertain if purification is complete,

since a great number ofthe steroids in canine urine are unidentified. So meas-

urements in crude urine may be helpful, but it is necessary to establish the nor-

mal ranges for each individual assay method, because, as is seen in the literatuIe,

figures for control measurements in crude urine can vary greatly depending on

the method used (Murphy et aI., 198l).

)l-u\ : } :

_:-<s aan influence rhe c"::.: associated rr irh rajs:,

- - i Conr ersel1,. rr ith de:i<_,_ :::.:rinine are to be erpe,

. :: :.ne elcretiol-ts than rr=

- , . mass in lr; peradren: :, .:-::ion as measured b1 ; r

-. -:nse. but that tl]e SuSP::

__. :: suppressibili§,(Fis :

::e ]. Range ofurine conis"_

reíadrenocorticism and p.r]\

, creatinine ratio is a sensitir:

::;ause the clinical ]Tanifesíai,

the diagnosis, The nur:

Plasma cortisol an,i -

::ternrined in samples obta:r

:lasma level varies episcldi.

:nd adrenocottical secIetiL,t

::: the 24-hour period rras si

:ire frequency of cortisol pe

:eriod in dogs, in contrast t.

:pisodes of secretiotr in rhe

;ircadian periodiciq, (Krieg,

cituitary-adrenocoft ical a.Ti

},s

,E

1500

1000

,,

(l

.!2

T 2010

0

500

0

Heallhy ccs Liver Diab€tes ccs Liver Diabetesdisoíd€ís mellitus disord€ís melhtus

ABFrg ,l Range ofserum alkaline phosphatase activity (ALP) and heat-Iesistant ALP

(StAP) activity in healthy dogs and dogs With hyperadrenocorticism (CCS), liver diseases

and diabetes mellitus, A: Mean and range ofALP in healthy dogs and dogs with hyperad-

renocorticism, liver disorders and diabetes mellitus. B: Mean and range of SIAP activityin the same groups as descrjbed in graph A. Mean and range ofthe percentage of total se-

rum alkaline phosphatase activity which is made up of the steroid-induced íiaction wele690Á,9 l00yo wh\ch illustrates very well that, however, slAP is not a specific test but a

sensitiye indicatoí of hyperadrenocorticism in dogs

By measuring cortisol in morning urine, an integration of tlre productionover a period of about 8 h is achieved, thereby adjusting for the wide and rapidfluctuations in plasma cortisol levels. The values are related to the creatinineconcentrations to correct for differences in urine concentrations. In man otlrer

Acta leletilarM Hlhgarica 46, l998

c/c ratio

)st 01'

llrichLlreS,

pur-lL hlrlete.

leas-nor-ture,g oll

DIACNOSlS OF HYPERADRENOCORTICISM IN DOGS

: :<]ses can influence the cortisol and creatinine excretions, Acromegaly in man

::en associated with raised urinary excretion of cortisol (Lindholm et al.,- -S t. Conversely, with decreased renal function, reduced clearances of cortiso|

,-: ;reatinine are to be expected. Dogs with hyperadrenocorticism had lower_,.::il]il]e excletions than the control dogs, which can be ascribed to tlie loss of-,.;le lnass irr lryperadretrocotticism, lt lnay even happen tlrat the cofticoid. - :uction as measured by c/c ratios is jLrst arourrd the upper limit of the refer-:, _3 range, but that the suspicion of tlre disease especially arises because of the

. _. of suppressibility (Fi8. 2).

12a

140

100

2010

0

]s

l-

Heallhy

|rg 2 Range ofurine cortisol:cIeatinine (c/c) ratio in healthy dogs and dogs with hy-peradrenocorticism and polyuria/polydipsia (PU/PD), Measurement of urine corti-

, ] ]:creatinine ratio is a sensitive test for hyperadrenocorticism (ccs) but it is not specific:ecause the clinical manifestation of polyuria./polydipsia due to othel disorders disturbs

the diagnosis. The number ofdogs in each group is slrown in parentheses

plasma cortisol and adrenal steroids. plasma concentfatioíls of cortisol:etennined in samples obtained from normal dogs at 20-min intervals show that

:lasma level varies episodically, wlrich presurrrably reflects episodic pituitary_lnd adrenocortical secretion. Tlre average number of cortisol secretory episodes:rl the 24-1rour period was similar to tlre number slrown to occur in humans but:he frequency of cortisol peaks was equally distributed throughout the 24Jlourperiod in dogs, in contrast to humans, who slrowed an increase in the number of!,pisodes of secretion in tlre early morning hours, coincident with the zenith in;ircadian periodicity (Krieger et al,, l971). Considerable individua| variability inp itLl itary-adrenocorlical activity was apparent in the dog, like in rats, some of

c/c ratio

(35}

m

lz2\ l§n m

Ácla Lblelinalia Hunganca J6, l995

THURoczY et al.

which could be explained on the basis of a sex difference, in that mean levelsand episodic activity of coíisol were generally greater in females. Because ofthe circadian rhl,thm and more or less sex differentiation, a 5-10 times higherbasal cortisol level can be found early in the aftemoon or in the evening than insamples obtained from 7:00 a,m, to l0:00 a.m., therefore basal plasma cortisoldetermination has little diagnostic value.

The plasma concentration of D}IA in sexually mature domestic animals isan order of magnitude lower than the normal adult values in man (normal range:

200-800 pgldl), A rise in DHA levels after sexual maturation, as is characteris-tic of adrenarche in man, is observed only in the rabbit and dog, and only in the

dog is there an accompanying rise in Aa-A as in man. Since DHAS levels wereundetectable or around the minimum level of detection in dog, it is not possibleto rule out increased DFIAS levels after sexual maturation.

In normal dogs, plasma 11-OHCS values are between 1.3-9.7 pgl100 mL,In about 50 per cent of cases the basal values in dogs with hyperadrenocorticismare in a similar range (Mulnix et aI., l976).

Plasma adrenocorticotropic hormone (ÁCTH) and a-ntelanocyle-stimulating hormone (u-MSH). ACTH is cleaved in the anterior lobe from a

large precursor molecule, pre-proopiomelanocortin (pre-POMC). This prohor-mone is ultimately processed into additional biologically active fragments.ACTH is composed of two final sequences: ct-MSH and corticotropin-like in-termediateJobe peptide (CLIP). Neither of these peptides is secreted as a sepa-rate hormone in men. Canine ACTH differs in the carboxy-terminal part of themolecule by only one amino acid from the ACTH of other species (Mol et al.,l99 l ).

In the pars intermedia of dogs there are two cell types that also can syn-thesise pre-POMC. One cell type is similar to the corticotropic cells of tlre ante-rior lobe, stained with anti-ACTH, and, in the other cell type, ACTH is cleavedinto ü-MSH and CLIP. High basal value of cx,-MSH indicates intermediate lobetumour origin, which tends to be DX resistant because of the absence of cortisolreceptors from the intermediate lobe. Basal plasma concentrations of ACTH indogs with pituitary dependent hyperadrenocorticism (PDH) were considerabIyhigher than in those with adrenocortical tumour (AT).

The mean baseline plasma ACTH concentration in healthy dogs is45 plmL (reference range: 2G-100 pg/ml), Endogenous ACTH concentrationless than 10 pglml in a dog with naturally occurring hyperadrenocorticism isstrongly suggestive of a functioning AT. About 35 per cent of dogs witlr PDHhave endogenous ACTH concentrations greater than l00 pglml- and 55 per centhave concentrations of45 to 100 pglml-.

D:].a), ::S :

-,_;.;]-, ígJ/s

:JTH stinu]ation r: sl ,

. . - riii of iarroseni. hr:,,. :;asnosis of sponrane: _

::-!.ie ánd not time consu,r

J.l_gs rr ith PDH ha\ e ;:]

, - l:.e hl perplastic adren3,

,,::_ ] ro s\nthesise e\cessl\

. --_. potential for an eraq:,Sr nthetic ACTH (Cünr

_ ,: : :]tlamuscularl; and s:=:

,_:,, 1. f and 4 h after st::r,

_ l3-ge) may be used, \cr::, :,. er. individua] labc]ratc:

, ,:;_-,l concentration, Resu -:!- : -,:renocorticism, making :..

1 ;. rr ith PDH aie not dislin.

' ::.Jogenous ACTH b) arr=,

_: -.:]!1cortical tissue. Despit:

- -.:; surface ACTH recep:c r,

_.,led by ACTH. Nlost c::. _TH stimulation test resulls

2000

1500

1000

500

0

baseline .

Healih,:.]

Fig, 3, Mean plasma conjsc, .

]dministration of synthetic AC -

\\ la

a)

,,1cla yelerinaria H n4arica 46, lg98

DIAGNOSIS OF HYPERÁDRENOCORTICISM IN DOGS

3 Dynamic tests

ACm simulation test, The ACTH stimulation test is most informative in::re diagnosis of iatrogenic hyperadrenocorticism but it has been commonIy used

--.r tlre diagnosis of spontaneous hyperadrenocorticism too. The test is safe, not.rpensive and not time consuming.

Dogs with PDH lrave adrenal hyperplasia secondary to chronic ACTH ex-:ess, The hyperplastic adrenals and functioning adrenocortical tumours have a

:.:pacity to synthesise excessive amounts of cortisol. Animals with PDH or AT.-.ar e the potential for an exaggerated response to ACTH.

Syrrthetic ACTH (Cortrosyn@, Organon, USA) 0.25 mg/dog is adminis-::red intramuscularly and samples are obtained before and at least l h but more.,,.actly l, 2 and 4 h after stimulation. Alternatively, 2.2 nJ1kg of body weight\CTH-gel may be used. Normal values must be established in each laboratory,:lrwever, individual laboratories obtain reasonably similar results for plasma:--,nisol concentration. Results were abnormal in 62 per cent of dogs with hy-:eradrenocorticism, making the test useful but not absolutely reliable. Results of_l,g5 with PDH aie not distinguished from those of dogs with AT. Suppression:; endogenous ACTH by adrenocortical tumours results in atrophy of all normal.lrellocortical tissue, Despite their autonomous function, these neoplastic cells-:tain surface ACTH receptors and intracellular pathways integral to a response: rused by ACTH, Most of the dogs with AT have abnormally exaggerated\CTH stimulation test results (Fig. 3).

)E

6EO

2000

1500

l000

500

0

7-T-

_--:post-stim, post-siim.

TllvIEHealthy dogs ccs

F€, J Mean plasma cortisol concentrations (+ 2 sD) determined before and 2 h after:Jlllinistration ofsynth€tic ACTH (cortrosyn@, organon, USA) in healthy dogs and dogs

with hyperadrenocorticism (CCS)

Acta relelinaria Hunqarica 46, 1998

l68 THURÓCZY et al

Dogs witlr clinical signs and routine laboratory test features of hyperad-renocorticism with a low baseline cortisol concentration and no response toACTH stimulation are likely to have iatrogenic Cushing's syndrome. No otherscreening test differentiates naturally occurring hyperadrenocorticism fromiatrogenic Cushing's syndrome.

Low-dose dexamethasone suppression (LDDS). Theorctícally, tlie admini-stration of relatively low dose, 0,01 mg DX/kg of body weight (Dexadresorreo,Intervet, The Netherlands) can inhibit pituitary secretion of ACTH and, in turn,decrease endogenous cortisol secretion for as long as 24 to 48 lt, Functionirrgadrenocortical tumours secrete excess cortisol autonomously, suppressing en-dogenous ACTH secretion, These tumours function independently of ACTHcontro|, Therefore, dexamethasone administration to these dogs ,,vould not affectthe plasma corlisol concentration at any time following administration. ChronicACTH secretion from functioning pituitary tumours causes adrenocortical hy-perplasia. The pituitary is somewhat resistant to the negative feedback effect ofcortisol, and a low dose of dexametlrasone causes variable degrees of suppres-sion of plasma cortisol shortly after administration but that suppression does notlast longer than 8 h (Fig. a).

2000

1500

1000

500

R.,p;,i.í.,r--l- :.rith1 d.,gs:=_:.:.iered in J:.: ::::_,] a.-rnl:al a --

_ :._.] c,_.ncen:t::

_:-:: i lrati..n C.-, .::: ralue and c:_ :::ism is the :.:: \:]i]etl]as!rne ti]

Dillirenti.i::; ]:i ií!-rn] AT :_ :,]]Lrl L. (] l 3

. :.. and (j t an S-

_ - aantratil-\n P-3- :a3 .-riteria \\ !,L..

Plasma c.,t-

_ :mol'L in 31 p,

- : ricisnt had a :-: .,:r -eiehr per cei

Hryh-tlt,,<:

.:]:noconical tu:,_:press _\CTH s

,:i er able to supp

: \CTH due t.-,

: _- se-depetldent n,

l\ kg of bodr rr,

-.: are defined as

:ls than 40 n m.,]

::rietrts nleet at ]

i,o i)HDDS car

:l.rrning urit]e sa

].n n\o consecLITI\

:dn inistration of: c ratio in healrh

_:ests hyperadrenj0% of ratios in i

In 55 per c

- 37 78) decreas,) l ,93), vhich sul

sUppressible non-suppressible

hypeíadrenocorticism

Fl& 4, Mean plasma cortisol concentations (+ 2 sD) before and following administationoflow-dose (0,01 mg/kg ofb, w.) dexamethasone (Dexadresoneo, InteíVet, The Nether-lands) (LDDS) in 78 dogs with hyperadrenocorticism, These dogs were diyided into twogroups on the basis of suppressibility of plasma cortisol by low-dose dexamethasone, In

48 out of the 78 dogs with hyperadrenocorticism (62%), 2 h after dexamethasone admini-stration the plasma cortisol concentration was less than 507o ofthe baseline concentra-tion. Plasma cortisol level was non-suppressible to 50% ofthe baseline concentration

in 30 dogs (38%)

Acla yelerinaria H ngarica 46, l998

169

Rapid dexamethasone clearance. Plasma dexamethasone concentrationsin healthy dogs persist for more than 12 h, Tlre dexamethasone clearance ratesare altered in dogs with hyperadrenocorticism, which may be estimated byplasma cortisol concentrations in 8-hour samples. Healthy dogs have a plasmacoftisol concentration less tlran 28 nrnol/L 8 h after low-dose dexamethasoneadministration, Cottisol concetrtrations betweerr 28 and 40 nmol/L have rro diag-nostic value and one must collect other information to determine if hyperadreno-cofticism is the correct diagriosis. Seventy-nine per cent of our patients cleardexamethasone fronr their plasma in less than 8 h.

Difíerentiation of PDH from AT by LDDS. The criteria to discriminatePDH from AT are (l) a 4-hour plasma cortisol concentration less than-i0 nmol/L, (2) a 4-hour plasma cortisol concentration less than 50% of basalleve|, and (3) an S-hour plasma cottisol concentration less than 50% of the basal.oncentration, plasma corlisol concentlations tl]at meet one or more of thesethree criteria would suggest the presence ofPDH,

plasma coftisol concentrations in the fourth lrour were less thatl]0 nmol/L in 32 per cent of dogs. Fifo,-eight per cent of dogs with hyperadreno-corticism had a 4-hour plasma coftisol concentration less than 50% of baselirie.Sixty-eight per cent ofour patients demonstrate at least one ofthese criteria,

High-dose dexamethasone suppression test (HDDS). The fi,rnction ofadrenocortical tumours is independent of pituitary ACTH, and they cornpletelystrppress ACTH secretion; therefore, regardless of the dose, dexamethasone isnever able to suppress the coftisol secretion, In contrast, clrronic hypersecretionof ACTH due to PDH tnay be suppressed by dexamethasone in a variable anddose-dependent manner. The test píotocol is similar to LDDS except that 0,1 mgDX/kg of body weight i.v. is used instead of 0,01 mg/kg. The suppression crite-ria are defined as a plasma coftisol concentration less than 50% of basal value orless than 40 nmollL 4 or 8 lr after administration of DX. Eighty per cent of ourpatients meet at |east one of these four criteria for suppression on the HDDS(Fis,5).

HDDS can be combined with measuremetlt of urinary c/c ratio frommonring urine samples on tlrree consecutive days. Basal samples are collectedon two consecutive days and the third sample is taken on the following day afteradrninistration of 0. 1 mg DX/kg of body weight. The upper limit of basal urinaryc/c ratio in healthy dogs is 10 x 10j. Exaggerated c/c ratio in basal samples sug-sests hyperadrenocofticism, and if the ratio in the third sample is lower than500% ofratios in basal samples, PDH is diagnosed.

In 55 per cent of our patients the basal urinary c/c ratio (mean + SD 3 9,66= 37,78) decreased at least 50%o after administration of dexamethasone (12.25 +1 l ,93), which suggests an ACTH dependency,

DIAGNOSIS OF HYPERADRENOCORTICISM IN DOGS

3sl features of hyperad-on and no response to:'s syndrome. No other:radrenocorticism from

;eoretically, tlre admirri-tr eight (Dexadresoneo,of áCTH and, in turn,: to 48 h. Functioning,.-usl1,, suppressing en-jependently of ACTH: do,ss would not affect: jninistration, chronic.ses adrenocortical hy-t:r e feedback effect of,.e degrees of suppres-:l suppression does not

._io§ ing administration. j:,]ten,et, The Nether-; ,.t ere divided into two]ie de\amethasone. In:.-\amethasone admini-:: b,aseline concentra-:5iljne concentlation

Acla I/eíerikaria Hungarica 46, l998

THURÓCZY et al,

6 15

Fig J, Results of high-dose dexamethasone supplession test, This graph illustrat€s that63 out of78 dogs With hyperadrenocorticism (8I%) showed suppression in the high-dosedexamethasone test (HDDS). Cortisol concentfation },,,as suppíessed by the LDDS test in47 dogs and there was no cortisol resPonse to the LDDS test in 16 dogs. A: cortisol de-

creases in both LDDS and HDDS tests; B: cortisol level is non-suppressible by low-dosebut suppressible by high-dose dexamethasone; c: no cortisol response to LDDS and

HDDS; D: cortisol level is suppressed at boü 4 and 8 h; E: suppression only at 4 h

In case of a non-suppressible urinary c/c ratio one has to speculate on dif-ferentiating AT from non-suppressible PDH due to a pituitary tumour arisingfrom the intermediate lobe.

CRH stimulation and metyrapone /e§t A single i.v. dose of 0.1 oCRHikgof body weight increases plasma ACTH and cortisol concentration in healthydogs, Dogs with hyperadrenocorticism due to PDH have an elevated plasmacortisol response to oCRH; in contrast, in dogs with AT there is no significantrise in plasma cortisol level.

Oral administration of 25 mg metyrapone/kg of body weight four times inevery 6 h is recommended for differentiating PDH from AT. Samples are col-lected before and 6 h after the final dose of metyrapone. The diagnosis of PDHcan be made if metyrapone causes a decrease in the plasma cortisol concentra-tion and a concomitant increase in plasma l l -desoxycortisol level. ]f both theplasma cortisol and l l -desoxycortisol concentrations decline after the admini-stration of metyrapone, the hyperadrenocorticism is due to AT.

50

Eo3E20z

]!l_-

10

Acla yelerinaria Huhgarlca 46, l99E

DIAGNOSIS OF HYPERADRENOCORTICISM IN DOCS

,': sualisation

Computed tomography. CT is a big step in the visualisation of pituitary or_ : ::al glands. Unfortunately, in 50 per cent of dogs with PDH, there are no or-

_, poorly visible microadenomas. CT scan is a non-invasive method but high, _-erses limit its use.

Scinligrapl,y. Radiocholesterol scintigraphy is a less frequently used tech-- ::e in the diagnosis of canine Cushing's syndrome; however, it has the same:,:. ]ntages in the localisation and characterisation of adrenocortical diseases as, :.Jmans. A few days after 15-20 MBq 13|I-iodomethyl- 19-norcholesterol ad-- -.:Stration repeated static images must be obtained. In our own experience, the- ::ral functional adrenal cortex cannot be visualised within the first two days, :.1gs. Bilateral uptake was found in nodular hyperplasia (5 cases), and unilat-.-:,. r isualisations proved to be adenomas (3 cases), which showed the clearest-:ging of adrenal glands (target/background íatio:3, in an average) on the,-,:..nd day after radiocholesterol application. Based on our own data (Thurócry.,. ll.. 1994) and on the only international reference (Mulnix et al., 1976) it cant stated that adrenocortical scintigraphy is a useful technique also in veterinary

=Cicine.

Acknowledgement

The present sfudy was supported in part by a gíant fiom üe Hungaíian scientific;:;earch Fund (OTKA F/13084).

References

., ,,rno, O. D,, Rossi, M. A., Contr€ras, L, N., Gomez, R, M., Galparcoro, G,, Cazado, E , Kral, M ,

Leber, B. and Aria§, D. (l985): Noctumal high-dose dexamethasone suppression test in theaetiological diagno§is of cushing's syndrome Acta Endocrinol, (copenhagen) l09, 158-162.

J-rler. P. W. P. and Bessr, G, M. (1968): Pituitaíy-adrenal function in severe depressive il|ness.Lancet l, 1234-1236.

...ntIeras, L, N,, Hane, s. and Tyrí€Ll, J. B. (l986)i Urinary coítisol in the assessm€nt of pituitary-adrenal function: Utility of 24-hour and spot determinations, J. Clin. Endocrinol, Metab,62,965_969.

_-_apo, L, (l979): Cushing's syndrome: A review ofdiagnostic tests. Metabolism 28, 955-977.-f.sai, R, K., Jialal, I., Naidoo, c., omar, M, A. and Joubert, S, M, (1987): Evaluation of the inte-

grated 3-hour p|asma cortisol concentration as a test for cushing's syndrome. S, AfL M9d.1.7l, l56_15,7.

l,aria, M, S., Kopelman, P. G., Besser, G. M. and Crossmarr, A, B, (1990): A comparison of theeffects ofhuman and ovine corticotropin-releasing hormone on the pituitary adrenal axis(abstract), Neuroendo crircl. 52, 4-ó.

rollenius, M,, Bradenbergeí, G, and H€itter, B, (1982): Diumal cortisol peaks and their relation-ships to meals. J, CIin, Endocrinol. Meíab.55,75H6l.

|1|

This graph illustrates thatuppression in the high-dosetressed by the LDDS test inin l6 do8s. A: cortisol de-r-suppressible by low-dosetI response to LDDS andsuppression only at 4 h

has to speculate on dif-lttuitary tumour arising

,, dose of 0.1 oCRH/kg]ncentration in healthy\e an elevated plasmarhere is no significant

il rreight four times in.{T. Samples are col_

The diagnosis of PDHma cortisoI concentra-:i;ol level. If both theline after the admini--{T.

Acla yelerinafía Hungalica 4ó, 1998

'IIlURoCZY et al

llanrct, P, Larochcllc, P, Franks, D J, carticr. P, and BoIté, D (l98?) cushing's s),ndlo e \\,ith

food-depcndent periodic hormonogcncsis clin Invest. Meal. l0, 530-533Ilanlng, R. V., Carlson, I. Fl,, Flood, c A. Hackett, R, J and Longcope, C (199la): Mctabolism

ofDHAS in normal rvonren ald rvomcn \vith high DHAS concentlations J, Clin Endocri-noi, Metab, 73, l2l0-12l5.

Ilaning. R. V. Flood. c. A,, Hackctt. R J,. Lo ghlin, J S, McClure, N and Longcope. c(199tó): Metabolic clcalancc rale of DIlAs. its mctabolism to testosterone, and its intra-lollicu]ar nretabollsnl 10 DFIA, androstcnedionc, tcstosterone and dihydrolestostclonc i/?

yi],o. J, cIin, EndocriDol, Mclab 72,1088-I095IIa},, R V,. Shapiro, B and Gross, M, D, (l996): Scintigraphic irnaging oflhc adr€nals and nculo-

cclodeonal tumors, In: Boles, M A. Dillehay, K- L et al (eds) Nuclear Medicine llenkinRE. Mosby, St Louis, pp. 855-876

Kal.c, T. B and Crapo, L (l990): Thc Cuslring's syndlome: An update on diagnostic tcsts, Ann,lntem Med ll2,434434.

Krieger. D.'t., Allen. W and Rizzo. F Fl P (l971): Charactelisation ofthe normal tcmporal pat_

tcln ofplasn]a corticostcroid levcIs, J, clin, Endocrinol Metab.32,266-284I-acrot\, A., Bolté, E, TrcmbIay, J and rramct, I' (l993), castrointestinal peptidcs and neoplasia:

llctopic castric Inhlbiloí),Polypeptidc (GIl') receptors in adíenal glands causing lood-dependent cushing's syndrome, N. Engl J, Mcd,4,307-308,

Ltddle. G W. (l960): l'ests of pitt]ilary-adrcnal sLrppresstvity in tl]c diagnosis ofCushing's s),n-

drome, J clin, Endocrinol, Mclab 20, l539-1560.Lindlrolnr, J, Kehlet, H, and Rushede, J (l978): Urinary cxcrction ofcoúisol inaclo egal),. J

Clin Endocrinol, Metab. 50

"796-1 98

Malchoff. C. D,. oíth, D N,, Abbol]d, C, Carney. J. A,_ Pairoleío, P, C and Carcy, R. M. (l988)lEctoPic AcTrI syndíome caused by a bronchial carcinoid tumor responsive to dcxamelha-sonc, nlet},raponc and cofticotropIn-rclcasing lactor. Am J. Med. 8,1,760-764,

Miller, D L- and Doppmarr. J. L, ( I99I ), |'etrosal sinus sampling: Tcchnlque and rational Radiol-ogy 178,37-47

MoI. J A . Van Mansfcld. D M,, K\rant. M, M., Van wolléren. M. and Rothl]izen. ]. ( l99l): 'lhe

gene cncoding proopiomclanocoúin iD thc dog. ^cta

l]ndocrinol. l25,77 83Mulnix_ J,

^., Van den l]rom, W E., Lubberink, A, A M. E., dcBrui;nc. J J. M, S and Riinberk_

A, (1976): Gantnta canlcra imaging of bilateral adrenocortical hl,pcrplasia and adrcnaI tu-moís ilr the dog Am. J, Vct Res 37, 1467-147l

Murphy, I}, E, P,, okouneff, l- M..Klein,G_P andNgo. S c. (l98l): Lack ofspccificity ofcorti-sol determinations in human uíinc, J Clin, EndocrinoL MeLab 53,90-99,

Nc\\ton, D R,, Dillon. W P. Norman, D, NeNton, T. H and Wilson, c, B. (l989), Gd-DlTA-enlranced MR imaging ofpituilary adenomas

^fu J Neurol l0,949-954

Nickelscn, ']',. l-issner, W arrd Schofflrng, K (1989)l Thc dcxomethasone suppíession test andIong-lerm contraceptivc trcalment| Measlrlement ol ÁCI'IJ or sallvary coíisol does notimpIove the leliability ofthc test, Exp Clin Endocrinol,94,275 280,

Nieman. L K, oldficld, íj. F]. Wcsley,R, Chlollsos. G P,, Loriaux, D. L and Cutter, C, l](l993): A simpliiicd morning ovine corticolropin-lclcasing hormone slinlulation tesl lolthc dilTercntial diagnosis of AcTIl-dcpendent cushing's syndromc. J. Clin Endocrinol,Mcíab 77,1308 1312

oldfield, D, M, Doppman, J. L.. Nicman. L, K, ch.ousos. G, P, Miller, D, L, and Katz, D A(l99I): Pelrosal sinus sampling with and \vithoUt corticotropin-releasing hormone for thediílbrentjal diagnosis ofCushing's syndrome, N. Engl, J Med,325,897-905.

Osella_ G-. ]'crzolo, M,, Borlefla, G,, Magro, G, ^li, ^,,

Piovesan, A. Paccolti. P. arrd Angcli. Á(1994): Endocrine evaluation of incidentall), discovercd adrenal masses (,ncIdcntalomas)J CIin. Endocrinol. Mctab 79. t532-1539

Icla lbleíhoíia H n?arica 1ó, I998

- Cushtng's syndrome rvithl0.5jO-j33,r.. C ( l99la): Metabolisn]:3nlrallons, J. cIin. Endocri_

.lrc. N, and Longcope, C]:jjlostcrone. and its intra-.]rd dihydrotcstosteronc i/l

:.lilhc adrenals and ncuro-\uclcar Medicinc, Henkin

,:: on diagnostic tests. rAl1n,

c; rhc normal temporal pat-j2,, 266-284::l peptides and Dcop]asia::3:3l _glands causing food-

::.:nosis of Cushing's sl,n-

DIAGNOSIS OF HYPERADRENOCOmIClSM lN DOGS

, G (1982): Investigation ofurinary steroid profiles as a diagnostic tnethod in Cushing's!\ndrome. clin. EndocrtDol (oxf,) 16,433439.

,_- _ C- and Silbcr, R H. (l950): A quantitativc color rcaction for cortisonc and lclatcdi -.2 l-dihydroxy-20-ketosteroids, J, Biol, chem, 185,20lJ01.

_ ]I . Besser. G, M,. J€ffcoat, W. J,, Goldjc, D, J and Marks, V. (l977)| Alcohol-inducad.scudo-Cushing's syldíome, Lancet l, 726-728

-j \l . Nieke, J . Krcstin, c P, saegcf. W, Allolio. B a d Winkclmann, W (1992): Prc-

.iinical cushing's syndrome in adrcnal "incidcntalomas": Comparison \{ith adrenal ctlsh-ing's syndrome J Clin, Endocrinol, Metab, 75,826-832

_,,i. A (l996): Adrenals In: Rijnbcík, A, (ed,) Clinical Dndocrinolog), of Dogs and calsKlu\tcí Acad Publ., Dordrecht. pp. 6l 94

:.-l, A, Der Kinderen, P J and Thijssen, J, H, Il (l968a), Investigations oflhe adrenocolLi-.al lunction ofnormal dogs J Endocrinol,4l,387-395

]:.i. ^.

Dcr Kinderen, P. J and Thlissen. J. Fl H, (l968ó): sponlallcous h},pcIadrenocolti-.isin in the dog, J, Endocrinol 4'l,,39'7406.

_ :eld. R, S, lJellman, L,, Roffivarg. |I , Weitznrann, E D,, Irukushlnla, D. K, and Galagl]cr.T, F, (l97l): Dehydroepiandroslerone is secleted episodically and s),nchroDlatousl},\!ithcorllsol by normal men, J, Clin, Endocrinol, Metab 33,87-95.

_ :ild, R, S,, Rosenberg, B. S., Fukushima. D, K, and Hellman, L (l975):24 hours secrclolypatteln of dchydroepiandrosterone and dchydroepiandrostcrone sulíáte J Clin, I]nclocri-

nol Metab.40,850 856,, _,l.L.J (t975)l 'I'rventy-four hour cortisol sccretoíy patterns in deprcssed and lnanic paticnts

Prog Brain Rcs, 42, 81-9l_:i. E T,(l968):

^ssessnent ol pituitaly-adrenal gland i'ullction in the dog,

^nr J Vct lics,

29. l7]-l80.::r. B, H,. Gritfrn, G. T and Melby, J, C (l983): Thc supcriority ofthc metyraponc test \crsus

|he high-dosc dexamethasone test in the diffolenLial diagnosis of cushing's syndrome Aml Med.74,65'1-662.

:j:.n. D H,. Stcvcnson. C 1', Da|akos, T. G, Nicholas, J, J, Dennick. L, G. and Fellernran. H( l969)| The diagnosis of hypercorticism: Biochemical cíiteria diffcrcntiatlng patients fíomlcan and obcsc normal subjects and from fcmales on oml contraceplivos. l. clin Endocri-nol Metab 29, l l91-12l l.

,.ie. E., Rothuizen, J,, DeBruiinc, J. J and Mol, J A (l989): Corticostcroid,induced alkalinephosphatase isoenzymc in the diagnosis ofcanine hypcrcorticisD Vct, Rec l25, l2 l1,

_róczy, J.. BaIogh, L,Iluszctlicza, Gy, Jánoki, G),, ^

and Magdus, M, (l994): Clinical dtag-

nosis of hypcradrenocorticism in dogs (in Hungarian) Kisállatorvoslás 4, 6-8.:..ia. E, saengcl, P., Pcterson, R, E, Rauh, W,, Gottesdiener, K., Levine, L S and NeN. lvl I

(l979): 6iydrocortisol excíetion in hypercortisolaemic states. J. Clin Dndocrinol Mctab48, 46,741l ,

, .]ker. M. S (l979): Screening for Cushing's syndronre using carly morning urinc sanrples, Ann-

Clin, Biochcm, 16, 965-969..:lson, R, B. Klcine, L, J,, Clalkc, T, J., |Icndricks, C, K arrd Grossmarr, M S (I967)i ltcsponsc

ofdogs lo corticotíopin nreasurcd by l7-hydroxycorlrcostcroid cxcrclion. Árn J Vel lles28,3l3 322.

:L]nisuI in acronregal),, J,

:nd Carey, R. M, (l988):: :jjponsive to dcxametha-

81.760_,764.::,,]Ue and lational Radlol-

l..ihuizen_ J. ( l99l): -I-hc

l]5.77-83: .] J \,l S. and l{ijnberk.,::rplasia and adrenal LU_

,:.i olspecifi city of corti__r.91-1-99

: B ( 1989): cd-DTPA-;-+ 9 95J.:,:j \upprcsslon tcst and::,:\ !r\ corlisoI does nol:!,] l, and cutlcl, G, B

::1]]a stimulation test lor::]: .l Clin EndocrinoL

::. D L and Katz, D A.,3,a]s]nc hormonc for the5. s9-_905_.:...iIi P and Angcli, A-:_rs(,s ( i ncidental omas)

AcIa yelelinarla Illl]]g()rtca Jó, ]998