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ng

COURSE CODE: VPT 304

COURSE TITLE: Clinical pathology
NUMBER OF UNITS: 3 Units
COURSE DURATION: Three hours per week

COURSE DETAILS:
COURSE DETAILS:

Course Coordinator: Dr. Omotainse DVM., MVSc., PhD

Email: [email protected]
Office Location: Department of Vet. Pathology, COLVET Building
Other Lecturers: Dr. Olaniyi, Moshood Olajire, DVM, MVSC &
Dr. Ajayi, Olusola Lawrence, DVM, MVSC

COURSE CONTENT:
Role of clinical chemistry and haematology in the management of clinical problems.
Determination of blood parameters including cell values (PCV, Hb content, RBC and
WBC counts) and plasma/serum concentrations of proteins, enzymes, sugar,
chemical ions and metabolites. Liver function tests, renal function tests.
Examination of urine for glucose, proteins, ketone bodies, blood cells, acidity and
bile pigments. Biopsy and exfoliative cytology procedures. Interpretation of clinical
pathology data.

COURSE REQUIREMENTS:
This is a compulsory course for all DVM students and attendance of at least 75% is required
to write the examination.

READING LIST:
1. Douglas J. Weiss and Jane K. Wardrop. Schalm’s Veterinary haematology. 6th Ed. USA.
Wiley-Blackwell, 2010.
2. Kaneko, J. J., Bruss, M. L. and Harvey J. W. Clinical biochemistry of domestic animals. 5th
Ed. Academic Press, New York. 1997.
3. Kerr Morag G. Veterinary Laboratory Medicine: Clinical biochemistry and haematology.
2nd Ed. London, Blackwell Science Ltd. 2002
4. Benjamin M. Maxine. Outline of Veterinary Clinical Pathology. 3 rd Ed. New Delhi.
Kalyani Publishers. 2007
5. Nduka Nsirim. Clinical biochemistry for students of pathology. Longman Nigeria Plc.
1999.

E
LECTURE NOTES
INTRODUCTION:
Haematology
Clinical chemistry
Clinical microbiology & Parasitology
Urinalysis
Cytology/histology (biopsy)
GENERAL
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In understanding the nature of a disease for effective treatment and control

measures to be adopted, pathology must go beyond postmortem diagnosis to
making use of changes in the structure and functions of organs/tissues in a living
animal. Clinical pathology provides evidence regarding the physiological alterations
resulting from pathological condition. Clinical tests make more meaning when
considered in relation to the history of the patient.
Clinical chemistry-Application of chemistry and it’s allied techniques for the
elucidation of disease, diagnosis and management of patient.
Blood chemistry is of great value in some disease in conformation of diagnosis,
prognosis and response to treatment {management of disease}. Interpretation of
such analysis is done by comparing results with normal range values of blood
constituents. Accuracy of such result depends on:
1) taking of appropriate samples in a proper manner.
2) availability of adequate information [history].
Clinical chemical analyses are mostly performed to determine parameters such as
blood Glucose, body electrolytes and other metabolites, enzymes, proteins [including
Ag & Ab] and renal functions.
Most clinical chemistry requires serum and occasionally plasma.

Blood for biochemical analysis will preferably require heparin as anticoagulant. This
is because heparin is a natural anticoagulant produced by the liver and prevents the
conversion of prothrombin to thrombin. Ammonium oxalate should not be used as
anticoagulant if blood non-protein nitrogen or urea is to be determined. The oxalate
and nitrates [eg Na-citrate] combine with Ca++ to prevent clotting. While
serum/plasma could be frozen for a limited period of time, depending on the required
analysis, whole blood should not be kept frozen to prevent lyses.
For general haematological examinations the anticoagulant of choice is EDTA
because it best preserves the cellular components and integrity as well as prevents
platelets aggregation. Haematological examination are mostly preformed in the
following areas:
1) Microscopic examination of unstained preparation
2) Microscopic examination of stained smears
3) Haemoglobin estimation
4) Packed cell volume determination
5) Red blood cell count
6) Total and differential leucocyte counts
7) Platelet count.
8) Clotting time and any other
BLOOD SAMPLE COLLECTION
Sample are generally taken for either haematological or biochemistry or serology
while haematological examination are performed with unclotted whole blood.
Serological examinations are made with serum from clotted blood, while collecting
blood, animals should not be excited unnecessarily. The site for blood sampling
depends on the purpose and volume of blood needed before taking blood, the site
should be sanitized by shaving [if necessary] and wiping the exposed skin with
alcohol or either and allowing it to dry.eg sites and animal species good for blood
sampling.
*Marginal ear vein-rodents and ruminants, pigs
*Jugular vein –for large quantities of blood –ruminants, horses, dogs
*subcutaneous abdominal vein –lactating cattle (anterior mammary vein)
*Middle coccygeal vein-cattle, pig
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*Cephalic (radial) vein-cat, dog

*Recurrent tarsal vein-cat, dog
*anterior vena cava-pig
*Cardiac puncture-Rodent
Samples should be collected in perfectly dry container to avoid
haemolysis. The superficial vein generally need to be occluded to distend
them by application of pressure with the fingers or a suitable tourniquet for
a brief time usually not long[2mins]. This should not be too tight.
Sampling requires sharp and correct size of needle. Needles are inserted
into the superficial vein at about 30 degrees to the skin.
Blood is withdrawn by gently applying negative pressure [traction] on the
plunger of the syringe. Blood is delivered into the container after removing
the needle. Selected anticoagulant is necessary in the container if
unclotted blood is required. Adequate mixing is ensured by gently inverting
the container several times immediately after sample is discharged into the
container to ensure proper and uniform mixing. The choice of
anticoagulant depends on the type of examination required.
Blood smears should be made as soon as possible, since anticoagulants
have effect on the morphology of the cells (rbc & wbc) when exposed to
them over a long time.
The rbc (biconcave cells) are produced in the bone marrow of adult
animals with the regulatory influence of erythropoietin, Cu, vit B12 and
proteins etc. Erythrocytes are generally/normally non-nucleated cells in the
mammals, nucleated in fish, reptiles & birds. The main function of the rbc
is to convey O2 to the cells/tissues by making use of haemoglobin
molecules it carries.
Haemoglobin is made up of 2 main units: Haeme and globin. The haeme
which isc a unit with iron carries oxygen to tissues. Pathology of the rbc
therefore is mainly in the area of production, morphology & number of the
cells and the nature of haemoglobin, ie. Haemoglobinopathy.
Erythropoiesis is a continuous process. The lifespan of a normal RBC is
about
140-150 Days in horse

80 Days in Cattle
52Days in Sheep
62-70 Days in Pigs
110-120 Days in Dogs
68-77 Days in Cats
Normally millions of RBC are being removed from circulation every minute due to old
age. An old RBC [senescent RBC]are removed from circulation either by in tissue eg
spleen or a small percentage get lysed in circulation. In both cases, the iron past of
the haeme is removed for reuse by the bone marrow. These normal produres could
also be disturbed in pathological cases.
Before being reused the iron moiety is locked in the macrophages as ferritin or
haemosiderin when need they are released into circulation back to the BM.The
remaining past of the haeme is converted in the liver to bilirubin.
Apart from the mature RBC which constitute about 95%/99% of the total erythrocytes
there are few mature cells in circulation. These are reticulocytes (non-nucleated) but
still with some RNA. Reticulocytes are better identified by special stains (supravital
stains):
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a) Brilliant cresyl blue in alcohol or saline for reticulocytes

b) New methylene blue
giving an orange coloured cell with purple network of RNA.
Normally, dogs , cats and pigs have little or reticulocytes in circulation; 0% in
ungulates. Presence of reticulocytes in noticeable number in peripheral
circulation is therefore a regenerative reaction of the bone marrow.

DETERMINATION OF PACKED CELL VOLUME [HAEMATOCRIT]

This is generally to determine the proportion in volume of the red blood cells
corpuscles in relation to the total volume of blood in circulation.
It provides a good evaluation of the RBC status ie. information about the
erythrocytes and haemoglobin in the circulatory system. This is b/c the
volume of the rbc in normal blood is directly proportional to their no. and to the
Hbg. Value.
General method:
A plane capillary tube is filled with anticoagulated blood eg. Blood with EDTA.
Alternatively, freshly collected blood could be filled into heparinized (heparin-
coated) capillary tubes, which are available commercially. Likewise
heparinized capillary tubes can be used to directly collect blood from the
animal eg. from the ear vein. The capillary tubes are filled to about  or  of
the length. The tubes are sealed at one end either with plastiseal or the use of
Bunsen burner. The tubes are arranged in a special microhaematocrit
centrifuge which is fitted with a head for carrying up to 24 capillary tubes. The
capillary tubes are arranged in a circular manner with the sealed end outward
(centrifugal) and the open end towards the centre (centripetal). The properly
covered m-centrifuge is set to rotate for 5 minutes at 12,000 rpm. PCV is read
by the use of special microhaematocrit Reader.
Buffy coat is a band of leucocytes and thrombocytes immediately above the
packed red cells.
Inferences:
-Anaemia-------Low PCV value
-Normal PCV
-Haemoconcentration-----High PCV value

Total Red Blood Cell Count.

Because of the large population of rbc in the blood, examination is only
possible after reasonable dilution has been made with suitable diluting fluid.

Examples of diluting fluids:

a. Hayem’s diluting fluid:
-1g. of Sodium chloride
-5g of Sodium sulphate
-0.5g of mercuric chloride
-200ml distilled water.
Filter if necessary.
b. Dacies’ fluid:
-99ml of 3% aq. Solution of Sodium citrate
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-1ml of 40% formaldehyde

Preferred for it keeps and preserves the cells better.
c. Physiological saline- where clumping occurs as a result of the above
fluids.

The basic equipment required in rbc total count are: microscope and
haemocytometer with the diluting pipettes. Blood sample is initially drawn into
the red dil. pipette held in horizontal position to the 0.5 mark on the stem. The
open end can be wiped off and can be used to withdraw the overdrawn blood
by the use of cotton wool or filter paper. In a vertical position in the dil. fluid,
draw the volume by gentle suction and rotation into the pipette beyond the
bulb to the 101mark . The pipette is further gently mixed for about 2 minutes
with the thumb and a finger at both ends in a horizontal position.
2 chambers separated by Central Moat
25 groups of 16 squares each =400 small squares.
It is recommended to select the 4 corner groups and the central group of 16
smallest squares, giving
1. Count only those erythrocytes which touch the left hand end and upper
lines of any square
2. Disregard those touching the right-hand and bottom lines.

R=Average of two(2) fields

Area of a smallest Sq=1/400mm2
Dept =1/10mm
Volume =1/4000mm3 for a Sq.
80 Squares =80/4000=1/50mm3
Dilution factor =200
Actual =200×50×R cells
=10,000R
eg R=(661+685)÷2=673

POLYCYTHAEMIA VERA
Primary –dehydration etc
Secondary – diseases-respiratory problem
ANAEMIA
Below normal range of the RBC and or haemoglobin value
-haemorrhage
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-haemolytic
-Dyshaemopoietic
-Hypoplastic
(*)-congestive heart failure
-chronic respiratory diseases
-Pulmonary &mediastinal neoplasia
There is tissue hypoxia which leads to increase in circulating RBC.

Evaluation of RBC parameter

Determination of Hb value:
1) Tallquist method: this is an indirect method. Comparing the colour of whole
blood with a colour standard. The method is not sensitive and could be of
upto 40% error
Here, a drop of blood on blotting paper is allowed to dry. The colour is
compared with a colour scale graduate upto 100% = 13.8 g/100ml. the
method is best used for screening purpose only
2) oxyHaemoglobin method : a direct method used to measure the
oxyhaemoglobin by high absorption with the help green filter in spencer
haemoglobinometer. A drop of blood is lysed in a glass chamber the RBC
are lysed by the used of applicator stick to obtain a clear fluid , which is
allowed unto the haemoglobinbinometer. The Hb is read off
3) Haematin method: acid haematin or Sahli’s method. Use of a special
haemoglobinometer and pipette. Mix fresh blood with N/10HCL in a pipette
to a standard level to for acid haematin. The colour is diluted to be at par
with colour of a standard tinted glass within 30min the content of the
pipette is there by recorded on the graduated scale.(defect in the
microvasculature through which RBC circulate)
4) Cyanmethaemoglobin method: 0.02ml of blood is added to modified
Drabkin’s solution 9put cyanide 0.05g , K-ferrocyanide 0.20g, dist H2O TO
1L) cyanmethaemoglobin is read in a photoelectric colorimeter with
suitable filter (yellow-Green)
5) Carboxyhaemoglobin

DETERMINATION OF MCV
Mean corpuscular volume. This is the cubical volume of the erythrocyte
MCV=(PCV×10)/RBC Fl or
PVC/RBC x 10
MEAN CORPUSCULAR HAEMOGLOBIN (MCH)
The amount of haemogobin expressed in each erythrocyte
Hb/RBC×10 pg

MEAN CORPUSCULAR HAEMOGLOBIN CONCENTRATION (MCHC)

Hb/PVC *100

CLASSIFICATION OF ANAEMIA
Base on Aetiological, morphological, responsiveness.
a). Based on Aetiology
1) Haemorrhagic anaemia
-acute blood loss (profuse blood loss eg. traumatic cut);
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-chronic blood loss

*chronic haemorrhages
-GIT ulcers
-Enteritis
-Coccidiosis
-Neoplasm
-Haemophilia[dogs &foals]
-Surgical operation
-Hypersplenism

Causes
Vit C & K deficiency
Parasites eg. Haemonchus contortus, Hook worms, ectoparasites
Plant poisioning eg. warfarin, sweet clover, bracken fern in cattle
-Traumatic injuries
-Idiopathic thrombocytopenia purpura

2) Haemolytic anaemia- caused by lyses of RBC within the vessels

Causes-Intrinsic
-Extrinsic
Clinical Feactures:
Splenomegaly
Lymphnodepathy
Jaundice
Lethargy
Headache
I) Intrinsic causes
-RBC membrane defect eg. hereditary spherocytosis
-abnormal haemoglobinopathy eg. sickle cell
-here metabolic defect as in pyruvate kinase deficiency
II) Extrinsic causes-Acquired haemolytic anaemia eg. Idiopathic auto-immune
haemolytic anaemia
-Iso-immune hereditary disorders eg. haemolytic disease of the new
born.[piglets, foals, puppies, Man(children)]
-incompatible transfusion
III) Infections-eg Babesia sp, Anaplasmosis in ruminants, haemobatonellosis,
bacillary haemoglobinuria (Cl. haemolyticum) in cattle & horses
IV) Drugs, Chemical poisions-Cu in [sheeps], Pb, Phenothiezine, mercury in
ruminants and pigs

3) Dyshaemopoietic anaemia- selective depression of erythrogenesis.

Causes :
a)nutritional deficiency –Cu , Co, Fe, Protein, Vit. Deficiency
b)parasitic diseases eg some worm infestation
c)chronic infectious diseases eg. chronic infectious nephritis in dogs, FeLV,
Ehrlichiosis
d)hypothyroidism
e) Neoplasm
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4) Hypoplastic (or aplastic) anaemia

Due to generalized bone marrow depression affect all other cells and platelets
This can be caused by:
-plant food poisioning eg. bracken fern in cattle, trichloro-ethylene from soya
bean meal in calf
-irradiation
-sulphonamide poisoning.

Based on Morphological classification:

Based on the size of the RBC and the content of the haemoglobin eg.
a)macrocytic, normocytic ,microcytic
b)hypochromic or normochromic
combine a)&b)
-microcytic hypo chromic
-macrocytic normochromic(megaloblastic anaemia)
-macrocytic hypochromic

c) Regeneration or non-regenerative anaemia

regenerative anaemia are those that the bone marrow is responding
adequately(actively) by increasing the production and release of erythrocytes
into the circulation. Here one can see immature RBC in
circulation[reticulocytes]. Such anaemia is characterized by the presence of
polychromasia. Special stains will desmonstrate reticulocytosis.
Regenerative anaemia is a feature of haemorrhagic and haemolytic
anaemias.
Non-regenerative anaemia shows that there are problems with the BM which
could be either nutritional or toxic.

Morphology of RBC in stained Blood Smears

1) Polychromatophilic cells –are those that pick up uniformly blue stain with
Romanowsky stain or Wright stain. The presence of such RBC in a film is
referred to as polychromasia.

2) Erythroblasts –with nuclei (nucleated RBC). These cells will disappear

normally after few weeks of age in circulation

3) Cells with eccentrically placed inclusion bodies known as Howell-Jolly

bodies eg. in cats and horses. They are regratile bodies when stained
with NMB. These H-J bodies (nuclear reminants indicate chemical or drug
toxicity eg . phenothiazine in horse

4) Anisocytosis: this describes the presence of variation in the size of RBC

5) Poikilocytosis: this describes the presence of variation in the shape of the

RBC not due to handling or making of the smear
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6) Schistocytes-fragments of RBC eg. in DIC(disseminated intravascular

coagulation)
7) Spherocytes –small rounded cells that are deeply stained without central
parlar eg. in immune-mediated haemolytic anaemia.
8) Heinz-Bodies-pieces of oxidized haemoglobin appearing as protrusion
(nose-like projection) from the side of RBC. They appear as pale circular
structures common in haemolytic anaemia caused by toxic materials like
onion, benzocaine copper which inflict oxidative injury on the RBC
9) Distemper inclusions- viral nucleocapsids which stain pink or blue
10) Reticulocytes (inmatured RBC)will show strips network or short chains of
ribosomes in the RBC. The appear as pale yellow cell with basophilic
precipitates of RNA. Best viewed at 100X. increased reticulocyte-
reticulocytosis
11) Anisocytosis and poikilocytosis result from the marked presence of
macrocytic cells,spherocytes , schistocytes ,microcytes and acanthocytes.

12) Acanthrocytes- RBC with 2-10 blunt elongated fingerlike surface

projections. It is associated with haemangiosarcoma in the liver.

13) Elliptocytes – oval shaped RBC

14) Dacryocytes- tear drop shaped RBC
15) Stomatocytes –cup shaped RBC
16) ECCENTROCYTES (blister cells) –RBC with haemoglobin concentration
at one pole with an unstained area at the other end.eg. in haemolytic
diseases, membrane defect
17) Borr-cells(Echino elliptocytes)-elongated RBC with ruffled margins
18) Crenation : presence of RBC covered by short spiky surface projections.
Most are artifact and can be confused with ecanthocytes. It can be
differentiated from ture poikilocytes for it is uniformly affecting almost all
RBC in a given film area as opposed to scatted RBC on the blood film.
Staining of wet blood film will produce REFRACTORY BUBBLES on the
surface of the cells.

Parasite like haemobartonella should not be confused with stain

aggregates which are ppts. of stains along the sides of the RBCs. The
aggregate do not resemble any of the parasites and vary in size. They
appear as refractile(jewel) when focused at 100x
Haemolytic Anaemia:
Results from defect on the RBC (inherent & acquired) or defect in
microvasculature through which the RBCs circulate.
Intravascular haemolysis- within the vessels
Extravascular haemolysis- outside the vessels eg by macrophages in the
spleen & liver.
Some haemolysis occur both intravascularly and extravascularly.eg.
haemobartonellosis, Babesiosis, pyruvate kinase def. Pyruvate kinase(PK) is
an enzyme in the glycolytic pathway essential for the production of ATP. ATP
is essential to maintaining RBC membrane stability. PK def. is a hereditary
disease in Dogs.
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Microangiopathic haemolysis-mechanical intravascular sharing of RBC while

passing through abnormally tortuous capillary beds eg in glomerulonephritis,
DIC and haemangiosarcoma.

Turbulent flow of blood- large vessels eg in heartworm disease and

TRAUMATIC disruption of red cell in heart disease.

Some characterics features seen in Anaemias

-Usually accompanied by mild pancytopenia
-Anaemia appear generally as Normocytic Normochonic to macrocytic
normochronic
-presence of occasional megaloblasts in blood films
-presence of occasional giant but fully haemoglobinized RBC
-presence of occasional RBC with bizarre and or multiple nuclear fragments
(inclusion bodies)
Ineffective erythropoiesis[Maturation Defect Anaemia(MD)]
a) Nuclear defect (megaloblastic anaemia)
-Failure of the precursor nuclei to divide and mature at the same rate as the
cytoplasm.
Asynchrony of nuclear/cytoplasmic maturation.
c) Cytoplasmic defect
-iron defect
-lead defect
-Vit. B6
-Large cells with immature pale nuclei with irregular chromatin clumps
-cytoplasm is too haemoglobinized for the degree of nuclear maturaration
Cytoplasmic Defect: failure of haemoglobin formation. The RBC continues to
divide, in the presence of mature nucleus.
Hypercellular red cell Bone marrow (BM). Small metarubricytes.
The RBC continue to divide to smaller cells because they never acquire full
complement of haemoglobin.
Normal reticulocytes produce/release at slow rate.
Blood film=microcytic,hypochromic anaemia
b/c of small RBC with increased area of central pallor
-fragile RBC=poikilocytosis
- low polychromesia

Normocytic normochronic anaemia

1) Anaemia due to inflammatory disease and neoplasia. The anaemia is
accompanied with hyperplasia of the white blood cells. BM (bone marrow)
smear will be with erythroid hypoplasia, granulocytic hyperplasia.
2) Anaemia with selective Erythroid Hypoplasia
eg. -low erythropoietin production
-Hypothyroidism (reduced oxygen demand by peripheral tissue)
- Selective RBC precursor cells destruction as in toxic or immune-
mediated mechanism.
In general bone marrow hypolasia(aplastic anaemia)
-severe anaemia
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-severe leucopenia
- thrombocytopenia may be present or absent
Aetiology –toxic or immune-mediated destruction of precursor cells
-myelophthisic anaemias
Toxic materials:- infection agents-FeLV, ehrlichiosis
-Toxic chemicals- Estrogen , drugs
[griseofulvin in cat]
-Ionizing radiation

Myelophthisic anaemia (occupation of the Bone marrow (BM) by other

abnormal cells & connective tissues)
a) Neoplastic diseases
b) Non-neoplastic diseases

1) Most common are:

-haematopoietic
-lymphoid leukemia, lymphosarcoma
-Granulocytic leukemia.

2) myelofibrosis [conn. tissue replacement of BM]

by -estrogen toxicity
-ionizing radiation
Blood Films:
i-non regenerative anaemia
ii-presence of poikilocytes

Polycythaemic –increased in circulation of RBC is characterized by increase in PVC,

Hb, RBC values than normal range values. Polycythaemia can be:
a) Relative: When there is a decrease in plasma volume due to dehydration.
b) Transient: Here polycythaemia is due to excitement and is usually just for
about an hour after which RBC values return to normal.
c) Absolute: due to increased BM[bone marrow] production of RBC.

 VETERINARY CL INICAL PATHOLOG Y

 BY Dr. AJAYI, O.L.
 Veterinary Clinic al Bioc hemis try
 PREAMBLES
 Variables affec ting c hemis try test res ults
 Many factors other than disease influence the res ults of chemistry tests. Thes e
fac tors may be pre-analytic al, analytical and pos t-analytic al.
Preanalytic al variables are variables assoc iated with the patient, sample c ollec tion
and sample handling. Thes e generally affec t the composition of the body fluid
before analys is.
Analytical v ariables are fac tors which influence the analytic al procedure.
Post-analytical v ariables inv olv e the different ways data from the laboratory is
pres ented, stored and transferred to the clinician.
Whenev er poss ible, thes e v ariables s hould be controlled in order to minimize their
effect on test outcome.
 PRE-ANAL YTIC VARIABLES
 Preanalytic al variables affect the composition of the s ample (and henc e s ample
quality) before the sample is analyzed in the laboratory. Thes e v ariables are the
most i mportant and c ommon sources of error in laboratory analysis and are often
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ov erlooked when interpreting test data. Control of preanalytic al variability inv olv es
proper s ample collection, preparation and handling by the veterinarian or
technician. W hen such factors cannot be controlled, they must be recognized and
considered when evaluating results for dis ease.
 Preanalytic al variables inc lude biological (patient) and non-biological (s ample)
fac tors
 Preanalytic al variables contd.
 BIOLOGICAL OR PATIENT VARIABLES Biologic al variables are ass oc iated with
the patient. There are factors that are inherent to the patient, such as breed, age
and sex, which cannot be controlled, but mus t be considered when evaluating tes t
results . Other biologic al variables inv olv e those factors which can be c ontrolled by
you, the v eterinarian, when drawing the blood s ample, such as ens uring the
animal is fas ted for 12 hours before sample c ollec tion.
 Preanalytic al variables contd.
 INHERENT PATIENT OR BIOLOGICAL VARIABLES These variables are
inherent to the patient and cannot be controlled. They must be c ons idered when
interpreting tes t res ults.
 Species: There are species -specific differences in the s ourc e of analytes e.g.
alanine aminotransferase is a liv er s pec ific enzyme in s mall but not large animal
species.
 Breed: e.g. healthy Draught horses typically have higher creatine kinase
values than Thoroughbreds

 Preanalytic al variables contd.
 Age: Many chemistry analytes change with age, i.e. young animals e.g.
elev ated alkaline phos phatas e in all s pecies
 Gender: Physiologic al s tates ass oc iated with pregnanc y or lactation in female
animals can affect analyte.
 Controllable Biological or Patient Variables
 Standardization of collec tion techniques can minimi ze the effect of these variables
on chemistry res ults. If these variables are unav oidable or desirable (e.g. s ample
collec tion post-exercise to detect rhabdomyol ys is in hors es), they mus t be
considered during tes t interpretation.
 Recent food ingestion: This will produce a post-prandial Lipemia. Lipemia may
affect the analytical methods us ed to meas ure certain plasma c ons tituents, thus
producing inv alid results.
 Stress: Stress (secondary to animal handling or underlying disease) may hav e
profound effects on laboratory results, due to endogenous corticos teroid and/or
adrenaline release.
 Exerc is e: The effec t of ex erc ise on plasma c ons tituents is dependent on both the
species and the intens ity of the exercise. In general, blood samples should be
collec ted from animals prior to exercise.
 Controllable Biological or Patient Variables c ontd.
 Drugs : The tec hnique of drug adminis tration, e.g. intramusc ular, c an direc tly affec t
analyte results. Which analytes may be affected by intramus cular drug
administration?
Drugs can also interfere with measurement of the analyte. Drug interference can
be grouped into two general categories :
1)) Physiologic al (in vivo) effec ts of the drug or metabolites on the analyte to be
measured
2) In v itro effec ts due to some physic al or c hemic al property of the drug or its
metabolites, whic h interfere with the actual ass ay proc edure.
This markedl y interferes with both hematologic , c hemis try and urinalysis results.
Interference with chemistry tes ts is dependent on the method and instrumentation
used.
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 Non-biologic al or Sample Variables

 Non-biologic al variables involve sample c ollection, handling and transport to the
laboratory. All of these variables c an be c ontrolled to mini mize the effec t they hav e
on laboratory results.
 Sample Collection
 Stric t attention should be paid to sample c ollec tion to minimize artefac ts induc ed
from poor sample c ollec tion.
 Venipuncture: Clean venipunc ture is ess ential to minimize artefac tual c hanges in
the results . Poor venipunc ture tec hnique may cause hemolys is, whic h alters
chemistry res ults in a variety of ways .
 Anticoagulant: The preferred samples for c hemistry tes ts are heparinized plas ma
or s erum (no anticoagulant). Fluoride-oxalate anticoagulant c an be used for
glucose. Citrate and EDTA anticoagulants s hould not be used for chemistry tes ts .
 Sample Handling
 After sample collection, strict attention s hould be paid to sample handling to
minimize the effects of these variables on results .
 Separation: W henever poss ible, especially when there is to be a delay between
sample c ollection and submiss ion to a laboratory, s erum or plasma s hould be
separated from cells as soon as possible after sample c ollec tion.
 Labeling: All body fluid samples taken from a patient s hould be correctly labeled
with the patient name or identific ation and the type of specimen (e.g. s erum,
plasma, s ynov ial fluid, peritoneal fluid).
 Storage: Mos t v eterinarians do not have the adv antage of hav ing a c linic al
pathology laboratory within wal king distance of their c linic . There is usually at least
a 24-hour delay between sample c ollec tion and delivery to the laboratory under
the latter c ircums tances .
 ANALYTICAL VARIABLES
 Analytical v ariables affec t the procedure by whic h the analyte is measured by the
ins trument. Analytic al v ariables are c aus ed by features inherent to the s ample,
e.g. interferenc es suc h as lipemia, or by features inherent to the analyzer. The
latter are minimized by using quality assurance proc edures within the laboratory

 Interferences

 The degree to which endogenous s ubstances interfere with c hemis try analyzers
depends on the type of analyzer, the methods used to detec t the analytes and the
amount of interfering s ubs tances in the s ample (i.e. laboratory-s pecific).

 Caus es of interferences
 Lipemia: Lipemia is c aus ed by inc reas ed triglyc erides (as chylomicrons or v ery
low dens ity lipoproteins ). Lipemia interferes with chemis try tests by the following
mechanis ms .
1) Light scattering: Res ults in falsely inc reased absorbanc e readings of some
analytes, e.g. total bilirubin, hemoglobin.
2) Volume displac ement/solvent exc lus ion : This falsely dec reas es values of some
analytes, e.g. electrolytes (mostly s odium and c hloride, but also potass ium to a
lesser ex tent).
3) Hemolysis: Hemolysis of erythroc ytes is enhanc ed in the presenc e of lipemia.

 5) Water release: Release of red blood cell water dilutes analytes.

 Icterus : Bilirubin interference aris es from its spectral properties and its ability to
reac t c hemic ally with other reagents (res ulting in dec reased analyte v alues). This
particularly affec ts creatinine concentrations.
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
 Hemolysis : Hemolysis is usually an in v itro artefact due to poor venipunc ture
technique, lipemia, freezing of whole blood s amples , delayed separation of serum
or plas ma from cells , or delayed s ample submiss ion. Howev er, hemolysis c an
occ ur in v iv o with certain types of hemol ytic anemias
Hemolysis interferes with chemistry tes ts by the following mechanisms:
1) Increased absorbance: Released hemoglobin increases absorbance in the
hemoglobin s pec tral range.
2) Inhibition of reactions : Releas ed hemoglobin can directly inhibit chemical
reac tions.
3) Analyte release: Release of analytes found in high concentrations in red blood
cells will falsely elevating the values of these analytes .
4) Enzyme release: Releas e of enzymes which participate in chemical reactions ,
e.g. adenylate kinase

.
 Hyperproteinemia: Monoclonal immunoglobulins (paraproteins), particularly when
pres ent in high c onc entration, interfere with c hemis try analysis by the following
mechanis ms :
1) Hyperv isc os ity: This affects sample v olume and is dependent on the class of
immunoglobulin (whic h immunoglobulins produc e hyperv iscosity and why?).
2) Binding to analytes : Immunoglobulin binding to s ome analytes produc ing
inc reased or decreas ed analyte values, e.g. hyperphosphatemia has been
reported in a dog with chronic lymphocytic leukemia and an IgM monoclonal
gammopathy.
3) Volume displac ement: This has effects simi lar to lipemia.
 Drugs : There are a number of different methodology-related drug interferences
that ma y bias results. One of the mos t c ommon drug interferenc es seen in
veterinary medic ine is the artefactual elevation of chloride values in samples from
dogs on bromide therapy (an anticonvulsant). It is always wis e to inform the
laboratory of any medications in a particular ani mal, s o that drug influences on
chemistry tests can be minimized (but usually not eliminated).

 Quality Control, Test Validity and Referenc e Values
 Laboratory Quality Control
 Valid laboratory tes t results can be among the most v aluable c omponents of the
diagnostic process , likewise invalid test res ults c an be among the mos t
dangerous.
 The ess ence of a good quality control is to defect potential s ource of error in
laboratory v alves .
 A good quality control programme must inc lude the following:
 (1) Systematic and periodic monitoring of proper operation of equipment s uch as
spectrophotometer, electronic c ell counters e.t.c .
 (2) A s ys tem of monitoring reagents inventory to ensure that reagents are in this
their ex piration dates .
 (3) Use of control with kno wn ranges of acceptable results for each tes t
performed in the laboratory.
 (4) Written records to monitor each of the above components and document that
they are being executed.
 Quality control should also be a daily practice.

 Quality Control, Test Validity and Referenc e Values c ont.
 Laboratory works performed in private practices is rarely performed in s uc h an
optimal s etting; therefore, the likelihood of s erious error is muc h higher.
 II Validity of laboratory test results
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 (A). Assay v alidation

 Becaus e reagents and ins trument us ed to perform laboratory tes ts on animals
specimens are often marketed solely for tes ting human spec imens , one c annot
ass ume that a known procedure will produce v alid results on samples from other
species.
 Quality Control, Test Validity and Referenc e Values c ont.
 Typical c omponents of a v alidation proc edure inc lude assess ment of:
 (a) Analytic al s pecificity: Which is the ability of the assay to measure the analyte
in question in the presence of potentially interfering s ubs tances .
 (b) Analytic al s ensitiv ity: Which is the lower limit of detec tion of the analyte.
 © Accuracy: This is the degree of agreement between the res ults and s ome
standard or true value.
 (d) Prec is ion: The precision of a laboratory meth od is its reproduc ibility. A tes t
can have a high degree of precision but a low degree of accuracy. Factors
influencing prec ision are ass ay methodology, instrumentation and skill of the
technician.
 A c ommon means of measuring and reporting the precision of a quantitative
laboratory method is the coeffic ient of variation (cv ).
 The cv is express ed as a percentage and c an be determined by running the
same sample repeatedly. The mean and standard dev iation of the set of results is
calculated, and the cv is ex pressed as follows:
 Cv = 100 (stand dev iation ÷ mean)

Quality Control, Test Validity and Reference Values cont.

 Acc urac y: This is monitored in the following ways .
 (a) Regular use of control materials for which values hav e been determined in
outside reference laboratories.
 (b) Regular participation in laboratory profic iency programs in which the
laboratory period really receives s amples of an known c ompos ition to be assayed.


 (2) Prec is ion
 (a) Graphic plots and rule based fac ilitate the ev aluation of accurac y and
prec is ion us ing control data.
 III Referenc e v alues are usually a set of values from a group of normal
healthy animals which c onform to a group of stated, s elected and known criteria.
 Needed to provides a bas is for c omparison with values obtained from s ick
animals.
 Quality Control, Test Validity and Referenc e Values c ont.
 The stated selec tion criteria may inc lude.
 (1) Clear definition of the clinical parameters us ed to s elect a c linically normal or
health animal.
 (2) Population parameter.
i Species (PCV higher in dogs than c ats)
 ii Breed (Ac tiv e breeds have higher pcv ).
 iii Age (young animal hav e higher protein concentration and lymphoc yte
 iv Sex .

Quality Control, Test Validity and Reference Values cont.

 3) Env ironment and physiologic al conditions.
 (a) Diet (animals on high protein diet have higher BUN)
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 (b) Fasting (B/d glucose and bile acids are lower

 following fas ting)
 (c) Pregnancy
 (d) Level of excitement (pcv and lymphoc yte are
 higher in exerc ise c ats ).
 (e) Body c ondition (Creatinine is higher in well-muscled
 animals).
 (f) Altitude (RBC of cattle is higher at high altitude).
 (g) Medications (c orticosteroids the Neutrophil counts
 and ALP are higher)

 Quality Control, Test Validity and Referenc e Values c ont.
 (4) Specimen collection and handling
 (a) Collec tion site
 (b) Anticoagulant
 © Sampling time
 (d) Collections interval before testing. .
 (e) Storage condition.
 (5) Analytic al method (inc luding instrumentation reagents , spec ific
 calibration s tandard and calc ulation method.
 (6) Statistical method used to derive referenc e intervals.
 The more div erse the population parameters , the higher the reference
interval. e.g. A population of dogs of any age, seize or breed, fasted or nonfas ted
varying in health.
 Reference values s hould not be trans ferred from one laboratory to the other
unless analytic al method is the same.

Quality Control, Test Validity and Reference Values cont.

 Reference interv al determination
 Clinical Relevanc e of Tes t Res ults
- Test v alues that fall within the referenc e interval are considered negative for a
specified disease condition. If the dis ease condition is abs ent, it is a true negativ e
(TN), and if the disease is pres ent, it is false Negative (FN).
- Test v alues that fall outs ide the referenc e interval are considered abnormal
and pos itive for the dis eas e condition. If the diseas e is present it is a true pos itiv e
(TP) and if the disease is abs ent, it is false pos itiv e (FP).
 Test v ary in their FP and FN rates for various disease and are defined by their
sensitiv ity, spec ificity and predictive value for the partic ular disease.

Quality Control, Test Validity and Reference Values cont.

 Sens itivity
 Sens itivity is the frequenc y of a pos itiv e or abnormal tes t result (e.g. one outside
the referenc e interv al) when a disease is present. It is the ability of the test to
identify diseas e s ubjects.
 Sens itivity = [TP ÷ (TP + FN)] x 100
 The more sens itiv ity a tes t, the more the FP, and less the FN. Therefore, when
sensitiv ity increases, s pec ificity dec reas e. Tes ts are s eldom highly s ens itivity and
specific at the same time.
 Narrowing the Referenc e interval will increase sensitiv ity at the expense of
specific ity. (FPs are inc rease and FNs are decrease). Screaming tests need a
high degree of sensitiv ity bec omes FN res ults are more unacceptable than FP
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results .

 2. Specificity
 This is the frequency of a negativ e or normal tes t res ult when a diseas e is absent
(i.e. the perc entage of TN res ults ). It is the ability to identify patient, that do not
hav e a particular dis eas e.
 Spec ificity = [TP ÷ (TP + FN)] x 100
 Widening the reference interval will increas e spec ificity at the ex pense of
sensitiv ity (FPs are decreas e and FNs are increased).

 Predictive value of a pos itive tes t (P+ )

 PV+ is the % of patient with a positiv e or abnormal test result that are
dis eased.
 PV+ = [TP ÷ (TP + FP)] x 100
 Pv + is affected by s ens itiv ity, s pecificity and prev alenc e of the disease in question.
 The PV+ of a tests is higher when applied to a population with a high inc idenc e of
the dis eas e; a positiv e test is more likel y c orrec t. This occurs when a population
is selec ted because c linic al signs or physical examination sugges t a particular
dis ease. The us e of multiple tes ts will inc rease the PV+. Tes ts in series ↑s
the Pv +. Spec ificity is ↑ at the ex pense of s ens itivity becos there will be fewer FPs
but more FNs when compared to us ing s ingle test.

 Predictive value of a negative tes t (Pv-)

 PV- is the % of patients with a positive or abnormal tests res ults that are free of
dis ease.
 PV+ = [TN ÷ (TN + FN)] x 100
 Doing multiple tes ts in parallel (biochemical profiles or batteries of tes ts) inc reas e
the Pv-. Sens itivity is increased at the ex pensed s pec ificity. There are fewer FNs
but more FPs when compare with a single test. The greater the number of tes ts in
the profile the greater the c hance of FPs.

REFERENCE RANGES
 Reference ranges vary considerably from one laboratory to another, and are
dependent on the methodology and ins trumentation utilized. This is es pec ially true
for s erum enzymes, where methods may v ary in pH, temperature, specific
cofactors and substrate used in reaction. As a cons equence, publis hed "normal"
values may not be valid for res ults generated by your lab. Referenc e ranges
should be establis hed by eac h laboratory, as they are instrumentation and reagent
dependent. You should not compare one laboratory's results to another
laboratory's reference interv als .
Reference ranges are us ually determined from a population of healthy adult
animals. There are 2 general methods for determining reference ranges, based on
the dis tribution of the data from these healthy animals . The resulting range then
will inc lude 95% of normal s amples, regardless of the method. As a result, up to
5% of normal animals may fall slightly outside the reference range for a giv en test.

 When numerous tes ts are run on the s ame animal, the c hances of obtaining one
or more slightly "abnormal" res ults on an animal that is actually normal rises (p = 1
- 0.95). For 12 tes ts, p = 0.46; for 21 tes ts, p = 0.66.

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 Gaussian distribution: This is when the data is normally dis tributed, i.e. distributed
symmetrically around the mean as illustrated to the right. The reference range is
calculated as the mean ± S, whic h encompass es 95% of the obs erv ations in
healthy animals . The top 2.5% and bottom 2.5% res ults from healthy animals will
fall outside an established range.
 Non-Gaussian dis tribution: For data that is non-gauss ian (i.e. skewed), the data
can be mathematically transformed, e.g. to logarithms. If this yields a normal or
Gaussian distribution, the geometric mean ± 2 SD can be us ed for reference
range determination (geometric means are based on the log-transformed data).
Howev er, in most ins tances, percentiles are us ed to c reate referenc e ranges from
non-Gauss ian data.

 CHEMISTRY PATTERNS
 Chemis try tests c an be grouped together on the bas is of body system or
physiologic process. Grouping tes ts into common parameters is the bes t way to
interpret chemistry data as it enables pattern recognition. Patterns of change
within and among thes e groups can prov ide us eful diagnostic information about
dis ease inv olv ement of v arious organ s ys tems. Grouping tests together c an also
help you s elect certain tes ts to identify disease processes in c ertain body s ys tems.
Tes t selec tion is important if cost is a fac tor in laboratory testing.

 The following is one way of grouping routine chemis try tes ts:
 Elec trolytes: Sodium, potass ium and c hloride.
 Acid/base parameters: Bicarbonate, anion gap.
Note: Acid base status is dependent on electrolytes, so these should be
interpreted together.
 Minerals: Calc ium, phos phate and magnes ium.
Note: These are often influenced by kidney function, so kidney parameters s hould
be evaluated concurrently. Also, many bov ine practitioners order mineral and
electrolyte panels.
 Protein parameters: Total protein, albumin, globulin, A/G ratio.
 Kidney parameters: Urea nitrogen, Creatinine.
Note: Urine specific grav ity s hould be c onc urrently ev aluated when ass ess ing
renal function, es pecially on initial presentation of the animal. In addition, kidney
function affec ts proteins , minerals , elec trolytes and acid-base balanc e, as well as
hematopoiesis. (Can you think of how renal disease alters hematopoies is ?)
 Liv er parameters:
Hepatocellular leakage enzymes: Alanine aminotrans ferase (ALT), Aspartate
aminotrans ferase (AST), Sorbitol dehydrogenase (SDH), Lac tate dehydrogenas e
(LDH).
Cholestatic enzymes: Alkaline phosphatase (AP), gamma glutamyl trans ferase
(GGT).
Liv er func tion tes ts : Total bilirubin, direc t bilirubin, indirect bilirubin, bile acids,
ammonia.
 Panc reatic parameters: Amylase, lipase, trypsin-like immunoreactiv ity.
 Carbohydrate parameters: Gluc ose, fructosamine, glycos ylated hemoglobin.
 Lipid parameters: Cholesterol, triglycerides .
Note: Lipid metabolis m is altered by diseas e process es affecting the pancreas and
glucose homeos tas is .
 Muscle parameters: Creatine kinase (CK), AST, ALT, LDH.
 Iron parameters: Serum/plas ma iron, total iron binding c apacity (TIBC), % iron
saturation of trans ferrin.
 Elec trolytes
 In general, electrolyte lev els in blood are influenced by changes in free water and
by changes in elec trolytes themselves, namely rate of intake, excretion/loss , and
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translocation within the body. Transloc ation can occ ur v ia mov ement into or out of
cells or into specific fluid c ompartments, such as a distended abomas um.

Sodium
 Interpretation of Serum Sodium Res ults
 Na+ c oncentration is inex tric ably linked with ex trac ellular fluid (ECF)
concentration, therefore interpretation of s odium lev els s hould always inc lude
consideration of the hydration s tatus of the patient (and therefore, changes in free
water). Sodium is the major extracellular c ation and is a primary determinant of
plasma os molarity and ECF v olume. The body attempts to maintain a cons tant
ECF v olume, as major c hanges in ECF volume
 Sodium cont.
 can hav e profound effec ts on the cell. The kidney plays a critical role in
ma intenance of ECF v olume, v ia sodium and water retention. Regulation of body
water is acc omplished by monitoring of plas ma osmolality (determined primarily by
sodium c oncentration) and blood v olume. This is achiev ed by osmoreceptors and
baroreceptors
 Sodium cont.
 Os moreceptors sense c hanges in osmolality. With hyperos molality
(hypernatremia), os moreceptors s timulate v asopress in or ADH secretion from the
pituitary gland and s timulate thirs t. Thirst is stimulated by as little as a 1-2%
decreas e in osmolality. The end result is water retention by the kidney and
inc reased water intake. Increases in free water will thus reduc e s odium
concentration. Opposite c hanges occur with hypoos molality.
 Baroreceptors are sensitiv e to changes in effective circulating v olume (ECV). The
ECV is that part of the extracellular fluid that is in the arterial s ys tem and is
effectively perfusing the tiss ues . It us ually varies directly with ECF volume. W ith
hypov olemia (decreased ECV), baroreceptors stimulate the renin-angiotensin
system, the end result being mineralocortic oid (aldos terone) release from the
adrenal cortex . Aldosterone s timulates inc reased absorption of NaCl and
promotes the exc retion of potassium and hydrogen in the distal tubules of the
nephron. NaCl retention promotes water resorption, thus correc ting the
hypov olemia. Hypov olemia also stimulates thirs t (a dec rease in ECV of 7-10% is
required for thirs t s timulation). Opposite c hanges occur with hyperv olemia.

 Hyponatremia

Hyponatremia results from retention of free water or excess loss es of s odium from
the body. Hyponatremia us ually (but not always ) indic ates a hypos molal state. It is
important to correc t severe hyponatremia gradually to prev ent this fatal
complication.

Caus es of hyponatremia
 Pseudohyponatremia: Artefactual decreas es in sodium oc cur with flame
photometry or indirect potentiometry in hyperlipemic and hyperproteinemic s tates.
Sodium concentrations c an als o be reduc ed in hyperos molar s tates, suc h as
hyperglyc emia or mannitol therapy.
 Sodium cont.
 Gain of free water
1) Excessive water intake: This will result in increased GFR and decreas ed
sodium absorption with natriuresis. This occurs with psychogenic polydips ia (has
been reported in large breed dogs), the syndrome of inappropriate ADH releas e
(ADH release without appropriate osmotic or volemic stimuli - has been reported
in dogs s econdary to heartworm infec tion and neoplas ia), antidiuretics and
hypotonic fluid administration. In these situations, animals are normovolemic.
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2) Perceived decreased ECV: Inappropriate water retention occurs when the body
perc eives a dec reas e in ECV and s timulates non-os motic ADH release (often due
to hypoalbumine mia). This occ urs in congestive heart failure, liver dis ease,
nephrotic s yndrome and adv anced renal failure (in this condition, there are
reduced numbers of nephrons to appropriately excrete the exc ess water from
polydips ia). In thes e s ituations, animals are hypervolemic .

 Loss of s odium in exc ess of water (hypertonic fluid losses)
Sodium can be lost in excess of water v ia renal or non-renal mec hanisms. This
usually results in hypov olemia.
1) Renal losses: This occurs due to lack of aldosterone (necessary for s odium
absorption in DCT of the kidney), proximal renal tubule dys func tion (resulting in
reduced sodium abs orption) in renal disease (es pec ially in hors es and cattle),
osmotic losses due to polyuria (diabetes mellitus), and diuretic therapy. Cattle with
renal failure have a c ons is tent moderate to marked hyponatremia.
2) Non-renal losses : This inc ludes gas trointestinal losses (diarrhea in c attle), third
space losses (ruptured or obstructed urinary tract, pancreatitis, peritonitis ,
chylothorax) and cutaneous loss es (sweating in hors es).
Horses and cattle with s ev ere diarrhea are v ery li kely to be moderately or
markedly hyponatremic and dehydrated. Hyponatremia is ex acerbated by
decreas ed intake of sodium in this s ituation. Dogs and cats with v omiting and
diarrhea are less likely to be hyponatremic , unless there are other causes of
sodium loss .

 Common causes of hyponatremia in small animals include gas trointes tinal losses
of sodium, diabetes mellitus (although c oncurrent dehydration may normali ze
sodium v alues), congestive heart failure (with or without diuretics), third space
losses, addison's disease, hypotonic fluid adminis tration, liv er dis eas e and diuretic
administration. Common c auses in large animals include diarrhea, s weating
(hors es), drainage of fluid, and s equestration within third s pac es .

 Normonatremia
 Serum Na+ concentration within the reference range can still indicate an abnormal
state if body water is abnormally high or low.
 Animals that are normonatremic but dehydrated hav e proportional deficits in body
water and body Na+. Vomiting, diarrhea, and renal dis ease are c ommon
conditions in which normonatremia and dehydration are found.
 Normonatremic animals with increased ex tracellular fluid have inc reas ed total
body Na+.

 Hypernatremia
 This c an dev elop if water is los t in excess or if Na+ is ingested in exc ess of water.
The firs t mechanism is the most common one. Hypernatremia is always
ass oc iated with hyperos molality and results in CNS signs due to cellular
dehydration.

Caus es of hypernatremia
 Pseudohypernatremia: This occurs with dehydration.
 Water deficit
Animals are usually normov olemic.
1) Excessive water losses: Panting (hyperthermia, fev er, heat s troke) or losses
through the kidney from diabetes insipidus (lac k of ADH). Animals will not usually
dev elop hypernatremia in these situations unless access to water is restricted
concurrently.
2) Inadequate intake: Lac k of access to water, primary adips ia/hypodipsia
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
 Hypotonic fluid losses : These can be renal or non-renal. Renal loss es can occur
with osmotic or chemical diures is or renal failure (e.g. polyuric renal failure in
hors es and cattle). Non-renal loss es inc lude gas trointes tinal losses (v omiting,
diarrhea), third s pace losses , c utaneous losses (burns). Animals will not usually
dev elop hypernatremia in these situations unless access to water is restricted
concurrently.

 Salt gain
Inc reas ed sodium intake (with res tricted water access, e.g. salt pois oning in
calves), hypertonic fluid administration, and increas ed s odium retention by the
kidneys, such as in hyperaldos teronis m.
 Hypernatremia is relatively unc ommon in s mall animals . It c an be seen with
diabetes ins ipidus (if water deprived), panting and hyperaldosteronism. In large
animals, it is often due to water restriction and salt pois oning, water deprivation,
renal disease and hypertonic s aline administration
 Potassium

 Interpretation of Serum Potass ium Results

 Potassium is the major intracellular c ation (intracellular K+ concentration is
approximately 140 mEq/L) and is important for maintaining resting membrane
potential of c ells. 60-75% of total body potassium is found within musc le c ells, with
the remainder in bone. Only 5% of potassium is loc ated in the ECF, therefore
potass ium c onc entration in blood is not always a reflection of total body potassium
lev els . Plasma (ECF) K+ c onc entration is tightly regulated; fairly s mall c hanges
can have marked effects on organ func tion.
Regulation of potassium
Ingested K+ is abs orbed non-selectively in the stomac h and small intestine.
Regulation of plas ma K+ is by renal excretion and mov ement of K+ from
extracellular fluid to intrac ellular fluid. If these mechanisms are functioning
normally, the amount of K+ inges ted has little effect on plasma K+. Howev er, if
one or more of the regulatory mechanisms is faulty, then the amount of K+
ingested c an ex acerbate abnormalities in plasma K+.

 Urinary excretion of K+ is largely by sec retion of K+ into the urine by the dis tal
tubules. 70% of filtered K+ is abs orbed in the PCT of the kidney regardless of K+
balanc e. 20% is abs orbed in the ascending limb of the loop of Henle and the
remaining 10% is deliv ered to the dis tal nephron. Principle cells in the dis tal
nephron secrete K+ and absorb Na+ under the influence of aldosterone.
Intercalated c ells in the distal nephron abs orb K+ in exchange for H+. K+ is als o
excreted in the c olon, which is als o influenced by aldos terone.
Trans location of K+ is largely dependent on insulin and catec holamines. Shifts of
K+ in and out of cells can also occur with changes in the pH of ECF.
Sev ere abnormalities of plas ma K+ are life-threatening s ituations .

 Hypo kalemia
 Hypo kalemia inc reas es the resting membrane potential of c ells, resulting in
muscle weakness, impaired urine c oncentrating ability, polydipsia and arrythmias.
It is usually due to gas trointestinal or renal losses of potassium. Remember that
blood K+ values are not always a reflection of total body K+ stores ; K+ values can
be normal in blood, despite severe defic its in total body K+.

Caus es of hypokalemia
 Pseudohypokalemia: Sev ere lipemia will result in a mild hypokalemia.
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 Decreased intake: This occurs with anorexia in large animals, including hors es
(espec ially foals), camelids and cows. Decreased intake in s mall animals rarely
results in hypokalemia unless there are additional loss es of potass ium.
Hypo kalemia can be seen in c ats fed low K+ diets.
 Transcellular shifts : Shifting of K+ from ECF to ICF occ urs with metabolic alkalos is
resulting in alkalemia, insulin release or administration, and c atecholamine release
(from adrenaline stimulating beta2-adrenergic receptors and ac tiv ating the
sodium-potassium [Na/K] ATPase pump in muscle). Similarly,. endotoxemia may
als o result in hypokalemia becaus e endotoxins als o s timulate the Na/K ATPase
pump in mus cle cells .


 Inc reas ed loss
The potass ium defic it will be enhanced if intake of potass ium is decreas ed.
1) Gas trointes tinal losses: Causes include vomiting of gas tric contents (the loss of
chloride enhances K+ excretion in the kidneys , promoting the hypokale mia), v agal
indigestion, and diarrhea. Diarrhea in hors es and cattle often produces a
hypokale mia. Severe diarrhea and v omiting in dogs and cats c an also result in
hypokale mia. Saliv a is potassium-rich and disorders such as choke in hors es and
cattle can res ult in hypokale mia.
2) Third space losses/s eques tration: Acc umulation of fluid in body cav ities (e.g.
peritonitis ) or dis tended gas trointes tinal system (e.g. volvulus , ileus) can result in
hypokale mia. This ma y be dilutional from perceiv ed volume depletion due to
losses of fluid from the intravasc ular spac e (with secretion of ADH and stimulation
of water intake).


 3) Cutaneous losses: Sweating (hors es).
4) Renal losses: Renal losses of potassium can occur via several mec hanis ms ,
the main one being aldosterone, whic h s timulates s odium abs orption in exchange
for potassium ex cretion in the principle cells of the c ollec ting ducts . Aldos terone is
stimulated by the renin-angiotensin s ys tem in res ponse to hypovolemia and
decreas ed deliv ery of c hloride (hypoc hloremia) to the mac ula densa.. Potass ium
excretion is also enhanced by increased dis tal tubule flow rates (any c ause of
polyuria, e.g. glucosuria, post-obstruc tiv e diuresis ), increas ed lumen
electronegativity (high concentrations of unads orbable anions in the renal tubule
lumen, e.g. penic illin, ketones), inc reased dis tal deliv ery of sodium and metabolic
alkalosis.


 Hyperkalemia
 Hyperkalemia dec reas es the res ting membrane potential, predispos ing the cell to
exc itability. This results in cardiac arrythmias.

Caus es of hyperkalemia
Hyperkalemia is usually due to decreased renal excretion of potass ium, due to
renal failure, urinary tract obs truc tion or rupture, or hypoaldosteronism.
 Pseudohyperkalemia
1) Serum: Serum K+ is higher than plasma K+ due to release of K+ from platelets
during c lotting. The differenc e between s erum and plas ma K+ in dogs with normal
platelet c ounts can be as high as 0.6 mEq/L. This differenc e is greater with higher
platelet c ounts.
2) Hemolysis: This will increase K+ in animals with high K+ in their erythrocytes ,
inc luding horses, pigs , cattle and c ats . Remember that certain dog breeds have
high K+ in their mature red blood cells, suc h as Akitas and other Japanese
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breeds. All dogs have high K+ in their reticuloc ytes, so K+ can be artefactually
elev ated in hemolysed samples (or samples with delayed separation from c ells)
with high reticuloc yte counts from any dog breed.

3) Leukocytosis : Very high leukoc yte c ounts (> 100,000/uL) can result in
hyperkale mia due to leakage of intrac ellular K+ from cells.
4) Age: Potass ium is higher in foals < 5 months of age than adult horses . Foals <
1 week old hav e higher K+ than foals > 1 week old.
5) K+ EDTA: Contamination of s erum/plas ma sample with K+ EDTA will result in
very high (non-physiologic) K+ v alues (>20 mEq/L) as well as marked
hypoc alc emia and hypomagnesiumia (due to c helation).

 Inc reas ed intake: This is usually iatrogenic and does not usually res ult in
hyperkale mia if kidney function is normal, e.g. KCl administration.
 Transcellular shifts : Shifting of K+ from ICF to ECF occurs with tissue necrosis ,
exercise (this occurs especially in horses and is due to release of K+ from
muscles - K+ is a local vasodilator for mus cle cells ), uroperitoneum (es pecially in
foals), hypertonic ity (e.g. diabetes mellitus - occurs due to s olv ent drag) and
hyperc hloremic metabolic ac idosis. A high anion gap metabolic acidos is (titration
acidos is) does not usually result in hyperkalemia as the organic anion mov es into
the cell with H+. Hyperkalemia in animals with a titration acidosis is not due to
translocation, but due to dec reased renal excretion of K+.


 Inc reas ed intake: This is usually iatrogenic and does not usually res ult in
hyperkale mia if kidney function is normal, e.g. KCl administration.
 Transcellular shifts : Shifting of K+ from ICF to ECF occurs with tissue necrosis ,
exercise (this occurs especially in horses and is due to release of K+ from
muscles - K+ is a local vasodilator for mus cle cells ), uroperitoneum (es pecially in
foals), hypertonic ity (e.g. diabetes mellitus - occurs due to s olv ent drag) and
hyperc hloremic metabolic ac idosis. A high anion gap metabolic acidos is (titration
acidos is) does not usually result in hyperkalemia as the organic anion mov es into
the cell with H+. Hyperkalemia in animals with a titration acidosis is not due to
translocation, but due to dec reased renal excretion of K+.


 Inherited c aus es
Hyperkalemic polymyopathy of horses : This is due to a genetic defect in the alpha
subunit of the sodium c hannel of muscle c ells (the s odium channels remain
perpetually open) obs erv ed in Quarterhorses and other heavily muscled breeds
like Appaloos as and Paints . The condition appears to be clinically wors e in males .
It is characterized by intermittent episodes of mus cle fasc iculation and weakness
concurrent with increas es in serum K+ v alues.
 Drug-induced hyperkalemia: Trimethoprim induc es hyperkale mia by inhibiting
sodium resorption in the cortic al collec ting ducts of the kidney.

 Chloride

 Hypochloremia
 Hypoc hloremia is always ass oc iated with a metabolic alkalos is and often results
in a paradoxic ac iduria. The low Cl res ults in sodium av idity in the kidney. Sodium
is absorbed in exchange for H+ and K+ as Cl c annot be absorbed with the Na+ to
ma intain electroneutrality.
 Caus es of hypochloremia
 Loss of c hloride > s odium: Vomiting of gastric c ontents, ptyalis m and gas tric reflux
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in hors es, sweating (horses), diarrhea in horses (espec ially due to large intes tinal
problems as chloride is absorbed in the ileum and colon of horses), renal diseas e
(espec ially in c attle), administration of high Na+ fluids .
 Sequestration of chloride-rich fluids : Dis placed abomasum, v agus indigestion,
gastric rupture, gas tric dilation-volvulus , gastrointestinal ulcers (horses).


 Hyperchloremia: This is ass oc iated with a tendency towards acidos is (HCO3 loss).

Caus es of hyperchloremia
 Pseudohyperchloremia: Bro mide administration - meas ured as chloride by ion-
specific electrodes.
 Drug administration: diuretics , adminis tration of Cl-containing fluids .
 Chloride retention: Renal failure, renal tubular acidos is, Addison's disease,
diabetic ketoac idosis, c hronic respiratory alkal osis.

 Acid-base status
 In health, blood pH (whic h is taken as the same as ECF pH) is maintained within a
narrow range of approx imately 7.4 to 7.5. Traditional interpretation of acid-base
status involves the Henders on-Hass elbach equation, where pH is determined by
the ratio of bic arbonate to carbon dioxide.

The major extracellular buffer of acids in the body is bicarbonate followed by

plasma proteins and bone. Intrac ellular buffers include proteins, organic
phosphate, inorganic phosphate and hemoglobin (in erythroc ytes). Regulation of
acid-base inv olves c hemic al buffering with intra- and extra-cellular buffers, c ontrol
of partial press ure of carbon diox ide by altering respiration and control of
bic arbonate and hydrogen excretion by the kidneys .

 The kidney is c entral to ac id-bas e regulation. Filtered bic arbonate is absorbed in

the PCT of the kidney and regenerates the bicarbonate los t in buffering acids
produced by normal body metabolis m. Hydrogen is excreted by the PCT and DCT
of the kidney. Excretion of H+ by the DCT is dependent on sodium res orption and
exc hanges for K+.

There are four primary types of acid-base dis orders: metabolic ac idosis,
respiratory acidos is , metabolic alkalosis , and respiratory alkalosis . The majority of
patients with ac id-bas e imbalance hav e either metabolic acidos is or metabolic
alkalosis or a mix ed dis order of both.

Acidos is

 Acidos is can be primary metabolic (decreas ed HC03) or respiratory

(hyperc apnea) or secondary in c ompens ation for a primary alkalos is. Acidos is has
profound effects on the body, res ulting in arrythmias , decreased c ardiac output,
depress ion, and bone demineralization.
 Primary metabolic ac idosis: This can be due to loss of bicarbonate
(hyperc hloremic metabolic ac idosis ) or titration of bic arbonate (high anion gap
metabolic ac idosis).


 Primary respiratory acidos is : This is due to increas ed pCO2 from decreased
effective alv eolar hypoventilation. Any dis order interfering with normal alv eolar
ventilation c an produce a respiratory acidos is . The most common c auses are
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primary pulmonary disease, ranging from upper airway obstruction to pneumonia.

Diseases or drugs that inhibit the medullary respiratory center also produc e a
profound respiratory ac idosis, e.g. general anesthes ia.
 Alkalosis

 Alkalosis can be primary metabolic (inc reased HCO3) or res piratory (hypocapnea)
or s econdary in compensation for a primary ac idosis. Usually res piratory alkalos is
is the compensatory mechanism for a primary metabolic acidos is . Alkalosis results
in tetany and convulsions, weakness, polydipsia and polyuria.
 Primary metabolic alkalosis: This is due to loss of hydrogen (us ually with chloride)
from the gas trointestinal or urinary tracts . Hypoc hloremia is a cons istent feature
and indic ator of metabolic alkalosis. HCO3 is generated on an equimolar bas is to
the amount of hydrogen lost.

 Primary respiratory alkalosis : This is due to hyperv entilation and is associated with
decreas ed pCO2. Hyperv entilation is us ually sti mulated by hypoxia associated
with pulmonary dis ease, congestive heart failure, or sev ere anemia. Ps yc hogenic
dis turbances , neurologic disorders, or drugs that stimulate the medullary
respiratory center, will als o s timulate hyperventilation
 Compens ation
 In general, changes in bicarbonate produc e compens atory c hanges in carbon
dioxide and v ic e v ers a. Compensation caus es parallel c hanges in pCO2 and
bic arbonate.
 Primary metabolic ac idosis: The primary abnormalit y is a dec rease in HCO3. The
compensatory res ponse includes extarc ellular buffering by bic arbonate,
intracellular and bone buffering (phosphate, proteins, bone c arbonate), respiratory
compensation and renal hydrogen excretion Metabolic ac idosis s timulates central
and peripheral chemoreceptors, thus stimulating alveolar ventilation (and
producing a s econdary respiratory alkalosis or reduced pCO2), e.g. dogs with
lac tate ac idosis from hypov olemia often hyperventilate (called Kussmaul's
respiration).
 Decrease in pCO2 = 1.5 [HCO3) + 8

 Primary respiratory acidos is : The primary abnormality is an inc rease in pC02. The
compensatory res ponse is intracellular buffering of hydrogen (s uc h as by
hemoglobin) and renal retention of bicarbonate (a s econdary metabolic alkalosis),
whic h takes s everal days to occur.
 Primary metabolic alkalosis: The primary abnormality is an increased HCO3. This
is initially buffered by hydrogen from ex trac ellular (mostly) and intracellular buffers
(such as plas ma proteins and lac tate). Chemoreceptors in the res piratory center
sense the alkalosis and trigger hypoventilation, resulting in increased pC02 or a
compensatory res piratory ac idosis. Naturally, the ex tent of respiratory
compensation will be li mited by the dev elopment of hypoxia with continued
alv eolar hypoventilation. In addition to respiratory compensation, the kidneys
excrete the excess bicarbonate (due to increased filtered bic arbonate and by
active HCO3 secretion by a s ubpopulation of intercalated c ells in the c ollecting
tubules). However, this takes s ev eral days to occ ur.


 Primary respiratory alkalosis : The primary abnormality is a dec reas ed pC02. The
compensatory res ponse to a res piratory alkalos is is initially a release of hydrogen
from ex tra- and (mos tly) intrac ellular buffers . This is followed by reduced hydrogen
excretion (mostly as ammonium phos phate) by the kidneys. This results in
decreas ed plasma bicarbonate which is balanced by an increase in chloride (to
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ma intain electroneutrality), thus producing a sec ondary hyperchloremic metabolic

acidos is . The pH can rev ert to normal from c ompensation in chronic respiratory
alkalosis.
 Reme mber these rules for c ompensation:
 Compens ation does not produc e a normal pH (exc ept in a chronic respiratory
alkalosis, in which compens atory metabolic acidos is can c orrec t the pH).
 Ov erc ompensation does not occ ur.
 Sufficient time must elapse for compensation to reac h s teady-state, approximately
24 hours.

 Primary respiratory alkalosis : The primary abnormality is a dec reas ed pC02. The
compensatory res ponse to a res piratory alkalos is is initially a release of hydrogen
from ex tra- and (mos tly) intrac ellular buffers . This is followed by reduced hydrogen
excretion (mostly as ammonium phos phate) by the kidneys. This results in
decreas ed plasma bicarbonate which is balanced by an increase in chloride (to
ma intain electroneutrality), thus producing a sec ondary hyperchloremic metabolic
acidos is . The pH can rev ert to normal from c ompensation in chronic respiratory
alkalosis.
 Reme mber these rules for c ompensation:
 Compens ation does not produc e a normal pH (exc ept in a chronic respiratory
alkalosis, in which compens atory metabolic acidos is can c orrec t the pH).
 Ov erc ompensation does not occ ur.
 Sufficient time must elapse for compensation to reac h s teady-state, approximately
24 hours.
 Characteristic findings in the different primary acid-based disorders with
appropriate compensatory changes are illustrated in the table below.

 The following formulas can be us ed to determine if compens ation is occ urring in
an acid-base dis turbanc e (in these formulae, N = midpoint of the referenc e range;
obs = measured v alue):

For a primary metabolic disturbance, the expected res piratory c ompens ation is:
 pCO2(ex pec ted) = pCO2(N) + [(HCO3(obs ) - HCO3(N)) x 0.7] +/- X;

where X = 2 for metabolic alkalosis, and X = 3 for metabolic ac idosis


For a primary res piratory disturbance, the expec ted metabolic c ompens ation is:
 HCO3 (expected) = HCO3 (N) + [(pCO2 (obs ) - pCO2 (N)) x X];

where X = 0.15 for acute res piratory ac idosis, X = 0.35 for c hronic respiratory
acidos is ,
X = 0.25 for acute res piratory alkalos is, and X = 0.55 for chronic res piratory
alkalosis

 Mixed ac id-base disturbances
 These are quite common and can be detec ted by non-parallel c hanges in HCO3
and the anion gap, chloride and pCO2. The following features give you an
indication that a mixed ac id-bas e disturbance is present:
 The pH is normal but there is an abnormal pCO2 and/or bicarbonate. (Remember
that compens ation rarely res ults in a normal pH.)
 Changes in pCO2 and bicarbonate occur in opposing direc tions. (Remember that
with compensation, c hanges in pCO2 and bicarbonate parallel each other.)
 Change in pH is opposite to that predicted from the pCO2 and HCO3.
 Compens ation exc eeds upper or lower limits .

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
 High anion gap

In an uncomplic ated high anion gap acidos is , the change in AG is equiv alent to
the change in bic arbonate. If the change in anion gap is greater or less (us ually by
2:1) than the change in bicarbonate, a mix ed acid-base disturbanc e is present.
 If the decrease in bicarbonate is greater than the increase in anion gap, this
indicates that there is a mixed disturbance, with something lowering the
bic arbonate greater than expected. In this ins tance, this is c ompatible with a
mixed high anion gap and hyperchloremic (normal anion gap) acidos is,e.g. renal
failure, res olv ing diabetic ketoacidosis, diarrhea with a high anion gap acidos is
(e.g. lac tic ac idosis).

 If the decrease in bicarbonate is less than the inc reas e in anion gap, this indicates
that there is a mixed dis turbance, with s omething prev enting the bicarbonate from
being as low as it should be. This is compatible with a mixed high anion gap
acidos is and metabolic alkalosis, e.g.lactic acidos is with sequestration of chloride-
rich fluid, renal failure with v omiting/diuretics , v omiting and diarrhea/diabetic
ketoac idosis/lactic acidosis . In this c as e, the bicarbonate and chloride will be low
and the anion gap will be high.


 Normal anion gap acidosis or alkalos is

In an uncomplic ated normal anion gap ac idosis or a metabolic alkalosis, the

change in chloride is equivalent to the change in bicarbonate. If the change in
chloride is greater or less (usually by 2:1) than the c hange in bic arbonate, a mixed
acid-base disturbance is pres ent.
 If the decreas e in chloride is greater than the inc reas e in bicarbonate, this
indicates that there is a mixed disturbance, with something decreasing the
bic arbonate. In this ins tanc e, this is c ompatible with a mixed normal anion gap
acidos is and a metabolic alkalos is. This c an occur in renal failure with
vomiting/diuretics, vomiting and diarrhea, and liv er disease.


 If the increase in chloride is less than the decrease in bicarbonate, this indicates
that there is a mixed dis turbance, with s omething enhanc ing the decrease in
bic arbonate. This is compatible with a mixed high anion gap and normal anion gap
acidos is

 Summary
 High anion gap with c hange in anion gap > c hange in bicarbonate. This indicates:
 1) Mixed high anion gap metabolic ac idosis and metabolic alkalos is. Most
common c auses include renal failure and v omiting, renal failure and diuretics ,
diabetic ketoac idosis and v omiting, lac tic ac idosis and v omiting.
 2) Non ac idotic high anion gap with normal or inc reas ed bicarbonate. This occurs
in a pure metabolic alkalosis , c arbenic illin therapy and dehydration (increas ed
albumin).
 3) Mixed high anion gap metabolic ac idosis plus res piratory ac idosis, e.g.
cardiopulmonary arres t.
 High anion gap with c hange in anion gap < c hange in bicarbonate. This indicates:
 Mixed high anion gap and normal anion gap acidos is .
 High anion gap acidos is masked by low anion gap (decreased albumin,
paraproteins ).
 Combined high anion gap acidos is and c hronic respiratory alkalosis.
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 Anion Gap
 The anion gap (AG) is c alculated from measured res ults by the following formula:
 AG = (Na++K+)-(Cl-+ HCO3-)
 Na+ and K+ account for about 95% of total s erum cations while Cl- and HCO3
acc ount for about 85% of the total serum anions in a healthy indiv idual. The
"normal" anion gap is due to the presenc e of various unmeas ured anions, e.g.
plasma proteins, phosphate, and s ulfate. An increas ed anion gap indicates the
pres enc e of excess iv e amounts of one or more of these anions or of some other
anion.

The formula shown abov e is deriv ed from the following equations:

 To maintain elec tric al neutrality, anions must equal cations. This can be
expressed as
 Na+ +K+ +UC = Cl- +HCO3- +UA

 Unmeasured cations (UC): Calcium, magnesium, and gamma globulins. Thes e
are rarely ev er inc reas ed enough to decreas e the anion gap.
Unmeasured anions (UA) : Lactate, phosphate, sulfate, ketones, metabolites of
some pois ons, suc h as ethylene glyc ol, plasma proteins.
 Rearrangement of the equation abov e gives -
 UA-UC = (Na++K+)-(Cl-+HCO3-)
Anion gap = (UA-UC)
 Sinc e UC c annot c hange enough to affect the anion gap and s till be c ompatible
with life, UC can be dropped from the equation. Thus,
 Anion gap = UA = (Na++K+)-(Cl-+HCO3-)
 As c an be seen from thes e equations, the AG is a figure that indicates the amount
of unmeas ured anions in serum.

Inc reas ed anion gap

 Titration ac idosis: Mos t c ommon cause of an increas ed anion gap.
 Alkale mia: loss of protons from plasma proteins (unmeasured anions) inc reases
their negative charge. Alkalemia also stimulates lactic ac id production. The
inc rease in AG is v ery mild.
 Dehydration: W ill increase plasma protein concentration (espec ially albumin).
 Sodium containing drugs : Sodium salts , penic illin.
 Age: Anion gap is higher in foals than adult horses .
 Decreased cations : magnesium and calc ium. Hav e mini mal affec t on the anion
gap bec ause of their low c oncentrations .
 Decreased anion gap
 Acidemia: Caus es dissociation of protons from plas ma proteins, decreas ing their
negative c harge.
 Decreased albumin: A v ery common cause of a lower than ex pected or dec reas ed
anion gap.
 Ass ay artefacts: Artefac tually elev ated chloride, e.g. bromide therapy.
 Dilution: Dilutes plasma proteins .
 Inc reas ed unmeasured c ations : Calcium, mag nes ium, gamma globulins , lithium.
(Thes e rarely c aus e an increased anion gap as mos t increases are incompatible
with life. It is unusual to see a low anion gap in multiple myeloma.)
 Mineral group
 Calc ium-phosphate homeostasis involv es interrelated ac tions of parathyroid
hormone (PTH), v itamin D metabolites , and calc itonin on kidneys, bone, and
intestines.
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Parathyroid hormone
PTH is secreted by chief cells of the parathyroid gland and results in inc reas ed
calcium and decreased phosphate in serum. Sec retion is s timulated by decreased
ionized c alcium, dec reas ed calcitriol, magnesium, dopamine, PGE2. Secretion is
inhibited by increas ed ionized c alcium and increased calcitriol.
Parathyroid hormone has 2 primary sites of action, the kidney and the bone. In the
kidney, PTH enhanc es renal resorption of calcium in the distal tubules , c ollec ting
ducts and ascending limb of the loop of Henle and promotes excretion of
phosphate in the prox imal renal tubules. It also ac tiv ates alpha1-hydroxylase in
the kidney, which converts vitamin D to its ac tiv e form, 1,25 (OH)2D. Parathyroid
hormone ac ts on osteoblas ts in bone, stimulating secretion of osteoc las togenic
cytokines, IL-6, IL-11 and PGE2 .

 Parathyroid hormone related protein (PTHrP)

Parathyroid hormone related protein is produced by s ev eral different c ell types

inc luding lymphoc ytes, squamous epithelium, endocrine glands , bone, skeletal
and smooth musc le and the kidney. The prec is e role of the protein is not known,
but it is thought to be important for cartilage formation, mammary gland production
and mov ement of calcium across the placenta and mammary gland. It is not
inv olv ed in calcium homeostas is in physiologic s tates. PTHrP has a s imilar amino-
terminal end to PTH and binds to PTH rec eptors . Therefore, PTHrP has a s imilar
effect on calc ium and phos phate as PTH. PTHrP is s ecreted by apocrine anal sac
adenoc arc inomas, s ome lymphomas and s quamous c ell c arcinomas . It is
respons ible for the paraneoplas tic hyperc alc emia seen in these disorders . Specific
ass ays for PTHrP are av ailable.


 Vita min D

Vitamin D is inges ted as vitamin D3 or produced in the skin under the influenc e of
UV light. It is transported to the liv er v ia vitamin D binding proteins where it is
converted (with the enzyme 25 hydroxylas e) to calcidiol or 25(OH)D. This is then
transported to the kidney, where it is converted to its active form, c alc itriol or 1,25
(OH)2D by the enzyme, alpha1-hydroxylas e in the proximal renal tubules . This
enzyme is s timulated by PTH, decreased c alcium or phosphate. It is inhibited by
calcitriol, increased c alc ium or phosphate.
Calc itriol stimulates calc ium and phosphate absorption in the intestine. In the
kidney, c alcitriol promotes renal phosphate res orption in the prox imal convoluted
tubule and calcium res orption in the dis tal convoluted tubule. In bone, calcitriol
fac ilitates the action of PTH on os teoblasts . Calcitriol can be produc ed by tumor,
macrophages and lymphoma c ells, and is responsible for the hypercalcemia of
ma lignanc y seen in s ome dogs with lymphoma


 Calc itonin

Calc itonin is produced by parafollicular c ells in the thyroid gland (C c ells).

Production is s timulated by an increase in ionized c alc ium. beta-adrenergic
stimulation, dopamine, es trogen, gas trin and glucagon.
Calc itonin counters the c alcium- raising effects of v itamin D and PTH by inhibiting
osteolys is and stimulation of renal excretion of c alcium.
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 Calc ium (Total)


 Total s erum calc ium comprises three major forms: ionized calc ium (about 50% of
total), protein bound (about 40% of total), and c alcium c omplexed with anions
suc h as bic arbonate, c itrate, lactate, and phosphate (about 10% of total).
 Hypocalcemia

 Clinical s igns of hypocalcemia in dogs include musc le tremors, convuls ions,

ataxia, and weakness . In horses , hypocalcemia is associated with synchronous
diaphragmatic flutter and signs of tetany inc luding stilted gait, musc le tremors,
flared nostrils , inability to chew, recumbency, c onv ulsions , and c ardiac
arrhythmias. In cows, hypocalc emia is usually manifes ted as weakness and
recumbenc y. Signs of hypoc alcemia dev elop when ionized c alc ium is too low for
normal muscle and nerv e function. Bec aus e of factors that influence ionized and
protein-bound c alcium fractions , the total calcium res ult does not necessarily
correlate with ionized calcium and is not by itself alwa ys a reliable indic ator of
clinical hypoc alcemia.
A mild hypocalc emia in the presence of hypoalbuminemia usually does not
indicate a disorder of calc ium

 Caus es of hypocalcemia

 Spurious . Hypoc alcemia can be due to hypoalbuminemia.

Calc ium can be meas ured reliably in heparinized plas ma and the results are
comparable to serum Ca res ults. Howev er, exposure of blood to antic oagulants
suc h as EDTA, citrate, and oxalate (the anticoagulant in the sodium fluoride tube)
reduces c alc ium to an unmeas urable lev el. Since Ca < 2 mg/dl is not compatible
with life, ex pos ure to agents that chelate c alc ium are indic ated by s uc h a result.
 Hypoparathyroidism. This has been reported in dogs, c ats and one horse. Clinic al
signs inc lude s eizures, ataxia, and lens catarac ts . It is c haracterized by
hypoc alc emia, normal or increased phosphate and normal magnes ium. Low
concentration of parathyroid hormone confirms primary hypoparathyroidism.
 Nutritional s ec ondary hyperparathyroidis m. This is s o-c alled bran dis eas e of
hors es (it occ urs with all grain diets or grass diets high in oxalates) but can occur
in all species with excess phos phate, reduced calc ium or lac k of v itamin D in the
diet. Ionized calcium v alues are low, stimulating PTH with bone res orption.


 Renal secondary hyperparathyroidis m. This occurs especially in dogs , but also in
cats with c hronic renal failure. PTH is stimulated from low ionized c alcium (due to
exc ess phos phate), v itamin D production is impaired due to renal ins ufficienc y.
 Milk fev er. This is seen in highly producing dairy c ows and res ults in paresis.
They have low calcium and phosphate. Magnesium is often normal. Ec lamps ia
can also be seen in dogs, cats, ewes , sows, mares and goats and produces
tetany in thes e breeds.
 Pancreatitis. Mild hypocalcemia, usually without clinical signs referable to
hypoc alc emia, is fairly c ommon in ac ute pancreatitis. The mechanism is unknown.
 Idiopathic hypocalcemia in foals. This is s een in young foals, from 4 days to 5
weeks old. They display tachycardia, sweating, mus cle rigidity, recumbanc y,
seizures and opisthotonus .
 Hypercalcitonism: C c ell tumors in dogs, horses and cattle.
 Tox icosis: Sodium phos phate enemas, blister beetle (c anthradin) tox icosis in
hors es , ethylene glycol toxicity (hypocalcemia is a common finding in the
chemistry panel of dogs and c ats poisoned with antifreeze
 Hypercalcemia
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 Hypercalcemia is not common in any species but is enc ountered more often in
dogs and hors es than in cats and cows . Ev aluation of a patient with Ca result
above or at the top of the reference range should be done with cons ideration of
albumin c oncentration and evidence of acid-base imbalanc e

 Caus es of hypercalcemia
 Physiologic. Young, growing dogs , es pec ially large breeds, often have Ca s lightly
higher than the referenc e range for adult dogs.
 Primary hyperparathyroidis m. This has been reported in both dogs and cats and is
due to c hief c ell neoplasia (adenoma or carc inoma) or hyperplasia. In dogs, it is
familial in Keeshonds and inherited in German Shepherd dogs. Primary
hyperparathyroidism is diagnosed by identifying a parathyroid adenoma by
surgic al exploration and biopsy and/or by meas uring high or normal PTH
concentration in conjunction with high ionized calcium v alues .
 Humoral hypercalc emia of malignancy. Hyperc alc emia is a paraneoplastic
syndrome in domes tic animals and is a great tumor marker. Lymphoid neoplas ms
are the mos t c ommon of the tumors to caus e hypercalcemia. The s ec ond is
adenoc arc inoma of the apoc rine glands of the anal sac . Primary or metastatic
bone tumors occasionally cause hypercalcemia. In hors es , hypercalcemia has
been s een with lymphoma, ameloblastoma, gastric SCC and an adrenal c ortical
carcinoma. In most ins tances , the hypercalcemia is due to elaboration of PTHrP,
although other cytokines (IL-6, IL-1) and vitamin D are involved.


 Addison's disease. The hypercalcemia is thought to be due to enhanced
absorption of c alcium in the gastrointes tinal tract, hemoc onc entration, dec reas ed
GFR from v olume c ontraction and inc reased complex ing and protein binding of
calcium. Replacement therapy with c orticosteroids returns the calc ium to normal
within a few days .
 Vitamin D tox ic ity. Until recently, hyperv itaminos is D was a rare cause of
hyperc alc emia. Rodentic ides c ontaining cholecalciferol are now widely av ailable.
Ingestion of thes e rodenticides produc es marked hypercalcemia within 24 hours .
Other c auses of hyperv itaminosis D are exc ess iv e dietary s upplementation and
ingestion of plants whose leav es contain c holecalc iferol (Cestrus diurnum or
Solanum malacox ylon, and Tris etum flav esc ens).
 Chronic renal failure. Hyperc alc emia should be attributed to renal failure only after
other caus es of hypercalcemia have been c ons idered and ruled out.


 Measurement of intac t parathyroid hormone (iPTH), ionized calc ium (ICa), and 25-
hydrox yv itamin D can usually disc riminate between the v arious c auses of
hyperc alc emia in dogs . Guidelines for interpretation of these
 tes ts in c ombination are s hown in the table at right.

 Phos phate
 Phos phate is absorbed in the small intes tine (es pecially the jejunum) by both
paracellular and active transport. Absorption is decreased by low v itamin D, high
calcium and low phos phate lev els in the diet and other compounds suc h as iron
and aluminiu m.
 Hypophosphatemia
 Phos phate is an ess ential component of ATP, the energy s ourc e of the cell. Mild
to moderate decreases in phosphate are common and are of minimal s ignific ance.
Sev ere hypophosphatemia c an produce the following c linic al s igns:
 Hematologic effec ts: Hemolysis is the most sev ere. ATP is required for normal red
blood cell membrane integrity. This is an important complic ation (life-threatening)
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of therapy for diabetic mellitus . Hypophos phatemia als o decreases neutrophil

function and platelet survival.
 CNS: Low phos phate causes seizures , coma and atax ia.
 Muscular: Ileus in the gas trointes tinal system and c ardiomyopathy may res ult.
 Kidney: Metabolic acidosis due to impaired bicarbonate abs orption and calciuria
result (with bone lysis ).

 Caus es of hyperphosphatemia
 Laboratory error: This can be due to high bilirubin, mannitol, and oxalate, EDTA
and citrate anticoagulants.
 Decreased absorption
1) Postprandial alkaline tide
2) Enteral nutrition: Phos phate decreas es 12-24 hours after tube feeding in c ats
and ma y induce intravasc ular hemolys is.
3) Miscelllaneous: Malabsorption, v itamin D defic iency, Ph-binding antacids ,
starvation, vomiting and diarrhea.
 Transcellular shifting
1) Res piratory alkalos is: This causes a dec reas e in blood pCO2 which increases
intracellular pH. The increase in pH stimulates phosphofructokinas e which
enhanc es glycolysis, c aus ing phos phate to mov e into c ells to supply the enhanc ed
phosphorylation that results. Res piratory alkalos is occurs with hyperv entilation
suc h as sec ondary to sepsis , heat stroke, CNS problems, hepatic c oma, s alicylate
tox icity and fear.
2) Insulin or gluc ose adminis tration
3) Hypothermia: This res ults in low phosphate, high calcium, and glucose, low
potass ium, acidos is and azotemia.

 Inc reas ed renal loss
1) Hyperparathyroidis m, ps eudohyperparathyroidism: Phosphate may be normal
or elev ated if there is a concurrent dec rease in GFR.
2) Renal dis eas e: renal tubular ac idosis, c hronic renal failure in horses.
3) Osmotic diuresis : Polyuria from os motic diuresis will result in renal phosphate
losses, e.g. diabetes mellitus.
4) Inhibitors of renal resorption: Tumoral osteomalacia res ults when s ome tu mors ,
espec ially mesenchymal tumors, release inhibitors of phosphate reabsorption.
These inhibitors are c alled phosphatonin. Impaired renal phosphate absorption
als o occ urs with Cushings s yndrome, v olume ex pansion, drug administration
(diuretics, cortic osteroids, sodium bicarbonate), and metabolic acidos is .
 Unknown mec hanisms: Hepatic lipidosis in cats , ox alate toxicity in large animals.

 Hyperphosphatemia
 Spurious Hyperphosphatemia
1) Hemolysis: Hemolysis or prolonged c ontact of serum with c ells in the blood
sample c auses mov ement of phosphate from the red cells into s erum and c an
raise the Pi res ult.
2) Post-prandial: A mild inc rease occ urs after eating.
3) Monoclonal gammopathy: Hyperphosphatemia c an be obs erved in monoclonal
gammopathies , due to binding of phosphate to the monoclonal protein.
 Decreased exc retion
1) Dec reased GFR: This is the mos t common cause of hyperphosphatemia. Man y
animals that are azotemic are also hyperphosphatemic. Ac ute and severe
reduction in GFR, as in ac ute renal failure or sev ere hypovolemia, is more likely to
result in hyperphosphatemia than is c hronic renal failure.
2) Hypoparathyroidism: Phosphate is retained whilst calcium is los t in the urine
due to lac k of PTH.
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3) Acromegaly: Gro wth hormone promotes retention of phos phate.

4) Hyperthyroidis m: Phosphate is inc reased in up to 21% of hyperthyroid cats.


 Inc reas ed abs orption
1) Hyperv itaminosis D: This produc es hyperphosphatemia as well as
hyperc alc emia.
2) Increased intake: Ingestion of excess phosphate (nutritional
hyperparathyroidism) or adminis tration of phos phate c ontaining fluids or
compounds.
 Transcellular shifts
1) Acute tumor lysis s yndrome: This res ults in high phosphate, high potass ium,
high uric acid and low calc ium. Animals often die of ac ute oliguric renal failure.
2) Severe soft tiss ue trauma: This can also res ult in inc reased phosphate as
phosphate is higher intracellularly than extracellularly, e.g. rhabdomyolysis
 Urea nitrogen and Creatinine
 These tests are used as indic ators of glomerular filtration rate (GFR). Neither is
perfect in this regard, but they are clinically useful nonetheless .

Decreases in GFR are generally due to one of two main causes:

 decreas ed renal perfusion due to hypov olemia or cardiac dys func tion (prerenal
causes)
 loss of functional nephrons (renal causes)
 combination of the two above causes
 Azotemia
 Azotemia is defined as an inc rease in urea nitrogen (UN) and creatinine and can
result form a v ariety of disorders including, but not limited to, renal failure. Uremia
is the term for the c linic al syndrome of renal failure with azotemia and
mu ltis ys temic problems such as polyuria, polydipsia, mild non-regenerativ e
anemia (in chronic renal failure), vomiting, wei ght loss, depress ion, and other
sequelae of inadequate renal function.
 Azotemia c an be due to prerenal, renal or post-renal causes . Differentiation of the
causes of azotemia requires urinalysis (es pecially as sess ment of urine s pec ific
grav ity), evaluation of c linic al s igns and res ults of other diagnos tic tes ts (e.g.
radiographic evidence of urinary tract obs truc tion).
 Prerenal azotemia

Prerenal azotemia is due to a decrease in GFR from circulatory disturbances
causing decreas ed renal perfus ion, s uch as hypovolemia (s hoc k, hemorrhage,
Addison's disease, vomiting), c ardiac dis eas e or renal v as oc onstric tion. Prerenal
azotemia can us ually be dis tinguished from renal azote mia by clinical s igns
(ev idence of dehydration or hypovolemia), urinalys is (urine s hould be
concentrated, i.e. > 1.030 in the dog, > 1.035 in the cat, > 1.025 in large animals;
and there s hould be no other evidence of renal tubule dysfunction such as
proteinuria, cylindriuria) and res ponse to therapy. Urine specific grav ity may be
decreas ed (despite a prerenal azotemia) if there are other factors reduc ing the
concentrating ability of the kidney (s ee urine spec ific grav ity).

 Therefore, often a res ponse to therapy (fluid administration) is required to
differentiate between a primary renal and prerenal azotemia (the azotemia s hould
correct with appropriate fluid therapy within 24-48 hours in a pre-renal azotemia) .
Note that many causes of a prerenal azotemia will result in renal hypox ia and
ischemia. If this is s evere or chronic enough, a primary renal azotemia ma y result,
and ma y c o-ex is t with a renal azotemia.
As UN levels in blood are dependent on flow rate through the renal tubules
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(dec reased flow rate in prerenal azotemia enhances renal absorption of UN, and
inc reases UN lev els in blood), UN may inc reas e without any increase in creatinine
in early pre-renal azotemias .

 Renal azotemia

Renal azotemia results from a decreased GFR when more than 3/4 of the
nephrons are non-functional. Renal azotemia may be due to primary intrinsic renal
dis ease (glomerulonephritis, ethylene glyc ol tox icity) or may be s ec ondary to renal
ischemia from prerenal c aus es or from kidney damage from urinary trac t
obstruc tion (post-renal azotemia). Loss of 3/4 of kidney function usually follows
concentrating defects (requires loss of 2/3 of the kidney), therefore isosthenuric
urine (usg 1.008-1.012) is common in renal azotemia. In addition, there may be
other ev idenc e of renal tubular dys function in the urinalys is , such as proteinuria,
granular or cellular casts, and glucos uria without hyperglyc emia (thes e features
are not always pres ent in urine from animals with a renal azotemia)..

Azotemia with a urine s pec ific gravity less than those values s tated abov e is
pres umptiv e evidence of renal azotemia or renal failure UNLESS there is also
ev idence of other diseases or conditions affecting urine concentrating ability
independently of renal failure. The greatest diffic ulty in differentiating renal from
prerenal azotemia is encountered in those cases with a urine s pec ific gravity
greater than isosthenuric (1.012), but < 1.030 in the dog, < 1.035 in the cat and <
1.025 in large animals

 Note that in cats, primary glomerular diseas e may oc cur without loss of renal
concentrating ability (s o the c at may hav e renal azote mia with conc entrated urine).
In horses and cattle, inc reases in UN are modes t in renal azotemia due to
excretion of UN into the gas trointes tinal system (the urea is broken down into
amino acids in the cecum and rumen, res pec tiv ely). Therefore, creatinine is a
more reliable indicator of GFR in thes e species.
As mentioned abov e, a high anion gap metabolic ac idosis is c ommon in all
species with renal failure. Hypermagnesemia and hyperkalemia are features of
oliguric or anuric renal failure in all s pec ies
 Post-renal azotemia

 Post-renal azotemia res ults from obs truc tion (urolithiasis ) or rupture (uroabdomen)
of urinary outflow trac ts . This is bes t diagnosed by c linic al signs (e.g. frequent
attempts to urinate without success or pres enc e of peritoneal fluid due to
uroabdomen) and ancillary diagnos tic tests (e.g. inability to pass a urinary
catheter) as urine s pecific grav ity res ults are quite variable. Animals with post-
renal azotemia are markedly hyperkalemic and hypermagnesemic. Uroperitoneum
can be confirmed by co mparing the conc entration of creatinine in the fluid to that
in s erum or plasma; leakage of urine is indicated by a higher creatinine in fluid
than in serum. Pos t-renal azotemia c an result in primary renal azote mia (failure)
due to tubule dys function from impaired renal flow.

 Urea Nitrogen

 Measurement of urea c oncentration in s erum is included in chemistry profiles

ma inly to sc reen for decreas ed glomerular filtration rate (GFR). Urea
concentration is measured as urea nitrogen, and the test is us ually called blood
urea nitrogen (BUN) or serum urea nitrogen (SUN). Concentrations of UN are
expressed in mg/dL.

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
 Urea is s ynthes ized by hepatoc ytes from ammonia generated by c atabolism of
amino acids derived either from diges tion of proteins in the intes tines or from
endogenous tiss ue proteins. Urea is excreted by the kidneys, intestine (high in
hors es ), saliva and sweat. In ruminants , urea is excreted into the gastrointestinal
system where it is conv erted to amino acids and ammonia whic h are then used for
protein production (remember urea is added as a supplement to many bov ine
diets).
Conc entrations of UN are dependent upon:

 Hepatic urea produc tion: The rate of urea production is dependent on hepatic
function and digestion and catabolis m of protein, i.e. urea formation is dec reas ed
in liv er disease (e.g. portos ys temic shunts ) and increas ed with protein c atabolism
or increased protein digestion in the intestine.
 Renal tubular flow rate: Urea is freely filtered through the glomerulus and
passively diffuses out of the tubules at a rate dependent on flow rate through the
tubules; the remainder of the filtered urea is excreted in urine. At high flow rates,
approximately 40% of filtered urea is reabsorbed. At low flow rates , as happens in
hypov olemic indiv iduals, approximately 60% of filtered urea is reabs orbed and
added bac k to the blood urea conc entration. This explains the high UN levels seen
with dec reased GFR of any cause.

 Caus es of increas ed UN

 Inc reas ed protein catabolis m: Fev er, burns, corticos teroid administration,
starvation, ex erc ise.
 Inc reas ed protein digestion: Hemorrhage into the gas trointestinal system, high
protein diets.
 Decreased GFR: Due to pre-renal, renal or post-renal causes.
 Caus es of decreas ed UN

 Decreased protein intake or protein anabolis m: Dietary restriction of protein,

young animals (high anabolic rate).
 Inc reas ed excretion: Any cause of polyuria, e.g. hyperadrenocortic ism, diabetes
me llitus.
 Decreased production: Liv er disease.


 Creatinine
 Creatinine is produced as the result of normal mus cle metabolism.
 Phos phocreatine, an energy-s toring molecule in mus cle, undergoes s pontaneous
cyclization to form creatine and inorganic phos phorous.
 Creatine then decomposes to creatinine. Production of endogenous c reatinine is
quite s table.
 An additional and relativ ely minor source is creatinine ingested during
consumption of animal tis sue and absorbed from the intes tines.
 Creatinine is filtered freely through the glomerulus and is not reabsorbed in the
tubules.
 Therefore, creatinine is a more reliable measure of GFR, compared to UN, in all
species as it is not influenced by diet or protein catabolis m.

 Caus es of increas ed creatinine

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 Artifact: When meas ured by the Jaffe technique (whic h is based on color
production and is us ed by the chemistry analyzer at Cornell Univers ity), both
creatinine and non-creatinine chromogens reac t with the reagent. Non-creatinine
chromogens include ac etoacetate, gluc ose, v itamin C, uric acid, pyruv ate,
cephalos porins and amino ac ids . When pres ent in high conc entrations, these can
artefactually elev ate creatinine v alues .
 Physiologic: Higher in foals (up to 8 mg/dL; thought to be due to defec tiv e
placental transfer) and heavily musc led hors es (up to 2.5 mg/dL).
 Decreased GFR: Due to prerenal, renal or post-renal causes. In ruminants and
hors es , creatinine is the best indicator of GFR.
 Inc reas ed production: A mild increase (< 1 mg/dL) may be seen after ingestion of
a recent meat meal. Ac ute myos itis does not consistently increas e c reatinine itself
(although s ev ere myositis or myopathy can produc e renal azotemia from
myoglobinuric nephrosis).

 Caus es of decreas ed creatinine


 Artifact: W ith the Jaffe reac tion, severe icterus (total bilirubin > 10 mg/dL or an
icteric index > 10 units ) may artefactually dec rease c reatinine concentrations (this
is based on data in humans and may not occur in animals or in every icteric
animal). This effect can be minimized by utilizi ng a reaction blank.
 Decreased production: Loss of muscle mass , young puppies (low mus cle mass ).
Sev ere liver disease from cirrhos is may result in decreased c reatinine v alues from
decreas ed creatine production.
 Inc reas ed GFR: This occurs in animals with portos ys temic shunts and during
pregnancy (due to increas ed cardiac output).

 Total Protein


Refrac tometry: This method is us ed for es timating plasma protein (including
fibrinogen) in EDTA plasma. It measures the refractive index of a sample relativ e
to the refrac tiv e index of water.

Biuret Method: This is the colorimetric method used on the automated c hemis try
analyzer. It detec ts all proteins and is acc urate for the range of 1-10 g/dl.
Turbidometric methods: Quantitativ e of protein in CSF, urine and other low-protein
fluids requires more s ensitive tec hniques than either the Biuret or refrac tometer
method.
 Albu min


Albu min is a globular protein with a MW of 69,000 daltons.
 It is s ynthes ized in the liv er and c atabolized by all metabolic ally active tissues.
 Albu min makes a large c ontribution to plasma c olloid os motic pressure due to its
small size and abundanc e (35-50% of total plas ma proteins by weight).
 It also serves as a carrier protein for many insoluble organic substances (e.g.,
unconjugated bilirubin).

Hyperalbuminemia
 Ov erproduction of albumin is not known to occur.
 Relativ e: Hyperalbuminemia is a relativ e c hange seen with dehydration. Globulins
will als o increas e in this situation, resulting in hyperproteinemia with no change in
A:G ratio.
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 Drugs : Increases in albumin are reported in experimental s tudies in dogs

administered corticosteroids. It is not c lear if this is due to increased produc tion of
corticosteroids or dehydration sec ondary to free water losses from c orticos teroid-
induced polyuria.
 Laboratory error: Albumin v alues c an be artifactually elev ated in s everely lipemic
or hemolyzed samples , but this is analyzer- and method-dependent. Albumin is
als o higher in heparinized plasma than s erum (due to non-spec ificity of
bromcresol green which also binds to globulins, inc luding fibrinogen), howev er
newer proc edures hav e been developed to minimize this phenomenon.

 Hypoalbuminemia

 Physiologic: Exc essiv e fluid adminis tration (overdilution).

 Decreased production
1) Decreased produc tion c an occur if there are
 insuffic ient amino ac ids available for hepatic
 produc tion of albumin. This occ urs in c as es of c hronic
 s evere malnutrition due to dietary defic iency) or
 s tarv ation.
2) The liver is the main site of albumin production.
 Chronic hepatic dis eas e will result in
 hypoalbuminemia when there is a > 80% reduc tion in
 functional mass.



 3) Acute phase reac tions stimulate downregulation of albumin produc tion. An
acute phase reactant res ponse is initiated in respons e to trauma, inflammation,
neoplasia, etc and inv olv es releas e of cytokines (IL-1, IL-6, TNF) from
macrophages.
 These c ytokines ac t on regulatory elements in hepatocyte genes, resulting in
upregulation of transcription of acute phas e reactant proteins (fibrinogen, s erum
amyloid A, ceruloplasmin, haptoglobin) and downregulation of transc ription of
other proteins, inc luding albumin and transferrin (so-c alled "negativ e acute phase
reac tants").
 Inc reas ed degradation of albumin may also play a role in the hypoalbuminemia in
this reaction.
 In this case, the A:G is decreased due to the combination of low albumin and high
globulins .


 Inc reas ed loss of albumin
This occurs with the following:
1) Protein-los ing glomerulopathy: This c an result in nephrotic s yndrome which is
charac terized by proteinuria, hypoalbuminemia, hypercholes torelemia and edema.
In thes e c onditions , albumin is lost, but globulin levels are usually maintained,
resulting in a low A:G.
2) Severe hemorrhage: Both albumin and globulins are lost, resulting in a normal
A:G.
3) Protein-los ing enteropathies . In these conditions, albumin and globulins are
usually los t conc urrently, thereby maintaining a normal A:G.


 4) Severe ex udativ e dermatopathies. This may also ass oc iated with c onc ommitant
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albumin and globulin loss (A:G tends to remain normal).

 Sequestration: Hypoalbuminemia can be due to seques tration of albumin within
body c av ities, e.g. peritonitis .
 Catabolism: This is not a well-charac terized mechanism for low albumin.
Inc reas ed albumin c atabolism may occur with negativ e energy balance or protein
ma lnutrition (e.g. chronic infec tions, neoplasia, trauma) and , potentially, as part of
an acute phas e response (s ee dec reas ed production above).


 Globulins
 Globulins c an be div ided into three frac tions bas ed on their electrophoretic
mobil ity. Most of the alpha and beta globulins are synthesized by the liv er,
whereas gamma globulins are produced by lymphocytes and plas ma cells in
lymphoid tissue.
 Alpha globulins: consist of alpha-1 and alpha-2 globulins .
Alpha-1 globulins include alpha-1 antitrypsin, alpha-1 antichymotrypsin,
oros omucoid (acid glycoprotein), serum a myloid A, and alpha-1 lipoprotein (HDL).
Alpha-2 globulins include alpha-2 macroglobulin (protease inhibitor), haptoglobin
(binds free hemoglobin), protein C (inhibitor of ac tiv ated coagulation fac tors FVIII
and FV), ceruloplas min (carrier of copper) and alpha-2 lipoprotein (VLDL).
 Beta globulins : c ons ist of beta-1 and beta-2 globulins.
Beta-1 globulins include transferrin (binds iron) and hemopex in.
Beta-2 globulins include complement factors 3 and 4, C-reactive protein,
plasminogen, beta-2 lipoprotein (LDL), beta-2 mic roglobulin and s ome proportion
of IgA (es pec ially) and IgM. Fibrinogen also migrates in this region.

 Globulins c ontd.
 Gamma globulins: consists of the immunoglobulins: IgM, IgA, IgG.
 For the routine c hemis try profile, total globulins are calculated as follows:
 TP - albumin = globulin

Globulins c an als o be meas ured quantitiv ely and qualitatively with electrophoresis.
Radial immunodiffusion is used for accurate quantification of immunoglobulins and
has also replaced immunoelectophoresis for determining the immunoglobulin
comprising a monoc lonal gammopathy.
 Hypoglobulinemia

 Decreases in alpha and beta globulins are not signific ant. Decreas ed gamma
globulins are seen when there is a defic iency of immunoglobulins (dependent on
class of Ig inv olv ed and severity of the decrease). Radial immunodiffus ion (RID) is
the bes t method for pursuing thes e diagnoses.

Decreases in globulins of all fractions may be seen in protein-losing enteropathies,

exudative dermatopathies, and hemorrhage. Conc omitant loss of albumin in thes e
conditions tends to maintain a normal A:G ratio with a low total protein.
 Inherited hypogammaglobulinemia


A v ariety of inherited immunodeficient syndromes hav e been reported. Although

some inv olv e cell-mediated immunity (e.g. PSCID), they often have concurrent
gamma globulin deficienc ies due to impaired helper T cell func tion.
 Primary sev ere combined immunodefic iency: This has been reported in Arabians
hors es (full and crosses ). It is charac terized by a lymphopenia, dec reased IgM in a
pres uc kle foal, abs ent IgM and IgA pos t-suckling. IgM, IgG and IgA are all low
after 3 months of age as maternally-derived antibodies are degraded. Animals
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hav e thymic and lymph node atrophy and die at a young age (usually when
maternal antibodies dis appear) of opportunistic infections , e.g. Pneumoc ys tis
carinii, adenovirus, cryptosporidios is.

 Inherited hypogammaglobulinemia contd.
 Agammaglobuline mia: This has been reported in foals . They hav e no B c ells and
lac k Igs by 3 months of age. T cell func tion is normal as are lymphoc yte counts.
They die of repeated infec tions , with a poor respons e to therapy, by 12-18 months
of age.
 IgM deficienc y: Selective IgM defic iency has been reported in hors es (Arabians ,
Paso Fino, quarterhorses and thoroughbreds ) and Dobermans . Hors es us ually die
of fatal pneumonia, arthritis and enteritis. Dogs usually hav e no c linical s igns as
long as IgG and IgA levels are normal.
 IgA deficiency: This has been reported in v arious dog breeds , including Sharpeis ,
Beagles , and German Shepherd Dogs . They suffer from recurrent infections
inv olv ing the urinary tract, respiratory trac t, and s kin.
 Trans ient hypogammaglobulinemia: This has been reported in Arabian horses and
dogs. They hav e a delayed ons et of post-natal i mmunoglobulin s ynthesis and are
susceptible to adenov iral and bacterial infections.

 Acquired immunodeficiencies
 These are, by far, more common than inherited immunodeficienc ies .
 Failure of pass iv e trans fer (FPT): Animals are dependent upon inges tion of
colos trum for passive immunity as immunoglobulins do not c ross the plac enta as
they do in human beings . FPT res ults when neonates fail to suckle or if dams leak
colos trum pre-parturition. For diagnos is of FPT, determination of IgG is
recommended within 24 to 48 hours of birth.

 Infectious diseases
1) Viruses: Feline leukemia v irus and feline immunodefic iency v irus are known
causes for ac quired immunodefic iencies in c ats . Canine dis temper v irus causes
immunodefic iency in dogs. Bovine v iral diarrhea c aus es immunodefic iency in
cattle and Aleutian mink disease virus (a parvov irus) c auses immunosuppression
in ferrets.
2) Parasites : Tox oplas mosis and Theileria caus e immunodeficienc y. Generalized
infection with Demodex canis is often found in immunodeficient dogs , however it
ma y be a result of immunodeficiency and not its c ause. Eperythrozoon wenyonii
infection in c attle is ass oc iated with reduc ed humoral immunity.
3) J ohne's disease caus es dec reas ed T c ell function.
 Acquired immunodeficiencies contd.
 Neoplas ia: Lymphoma in cattle and horses is associated with
immunosuppress ion. Very low IgM lev els are often observ ed in horses with
lymphoma and c an be a valuable non-inv asive tumor marker if there is a high
clinical index of s uspic ion for lymphoma.
 Idiopathic : Idiopathic immunodeficienc y has been reported in young llamas with
failure to gain weight, ill-thrift and rec urrent infec tions. Many of thes e llamas hav e
concurrent Eperythrozoon infec tions .

 Hyperglobulinemia
 Inc reas es in total globulins c an res ult from increases in any or all of the frac tions
as determined by electrophores is.

Alpha globulins
 Acute phas e reactant respons e: This us ually res ults in increased alpha (espec ially
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alpha-2) globulins . Ac ute phase reactants are a divers e group of proteins that
inc rease in serum v ery rapidly (within 12-24 hours) following tiss ue injury of any
cause (inflammation, ac ute bacterial and v iral infections , necrosis, neoplas ia,
trauma). Rais ed serum lev els are the result of increased hepatic synthesis
mediated by cytokines (IL-1, IL-6, TNF). They als o tend to remain elev ated in
chronic inflammatory c onditions .
 Nephrotic syndrome: A dramatic increase in alpha-2 globulins is often seen (due
to VLDL and alpha-2 mac roglobulin).
 Drugs : In dogs, c orticosteroid administration results in an increas e in alpha-2
globulins .

 Hyperglobulinemia contd.
 Beta Globulins
 Inflammation (acute and chronic): increas ed beta globulins often accompanies
inc reases in gamma globulins (respons e to antigenic stimulation).
 Active liv er diseas e and s uppurative dermatopathies (both of which are assoc iated
with elevated IgM).
 Nephrotic syndrome (associated with an inc reas e in transferrin).

 Gamma Globulins


Inc reas es in this fraction occ ur mos t commonly in c onditions in whic h there is an
active immune respons e to antigenic s timulation us ually res ulting in a polyc lonal
gammopathy. Neoplas ms of immunoglobulin-producing cells (plasma c ells, B-
lymphocytes) c an als o be res ponsible for monoclonal increas es in this frac tion.
 Polyclonal gammopathy
This is s een as a broad-bas ed peak in the beta and/or gamma region. Some
common c auses include various c hronic inflammatory dis eas es (infec tious,
immune-mediated), liv er dis ease, FIP (alpha-2 globulins are often conc urrently
elev ated - see adjac ent ELP tracing), occult heartworm disease, and Ehrlichiosis .
Beta-gamma bridging occurs in disorders with increased IgA and IgM s uch as
lymphoma, heartworm disease and c hronic activ e hepatitis .

 Gamma Globulins contd.
 Monoc lonal gammopath y
This is s een as a s harp spike in the beta or gamma region. The peak can be
compared to the albumin peak - a monoclonal gammopathy has a peak as narrow
as that of albumin. Both neoplas tic and non-neoplas tic dis orders can produc e a
monoc lonal gammopath y.
1) Neoplasia: Multiple myeloma is the most common c aus e (producing an IgG or
IgA monoclonal). Other neoplastic dis orders ass oc iated with a monoc lonal
gammopathy inc lude lymphoma (IgM or IgG) and chronic lymphocytic leukemia
(usually IgG). Extramedullary plas macytomas are s olid tumors compos ed of
plasma c ells that are usually found in the skin of dogs . They hav e also been
reported in the gas trointes tinal tract and liver of cats and dogs . They can be
ass oc iated with a monoc lonal gammopathy, or ev en a biclonal gammopathy (if
there are multiple tumors).
 Gamma Globulins contd.
 An increase in IgM is called mac roglobulinemia. W aldens trom's
macroglobulinemia is a neoplas m of B-cells (lymphoma) that has a different
pres entation from multiple myeloma. Patients us ually hav e s plenomegaly and/or
hepatomegaly and lack os teolytic les ions. In contrast, multiple myeloma is a
dis order of plasma c ells that hav e undergone antigenic s timulation in peripheral
lymph nodes and then home in on the bone marrow (the marrow produces
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appropriate growth fac tors that s upport growth of myeloma c ells). Therefore,
myelo ma is charac terized as a bone marrow disorder, with osteolytic bone les ions
(in 50% of c anine cas es) and Bence-Jones proteinuria.

 Gamma Globulins contd.

 2) Non-neoplastic disorders: Monoclonal gammopathies (usually IgG) hav e been
reported with occult heartworm disease, FIPV (rarely), Ehrlic hia canis ,
lymphoplasmac ytic enteritis, lymphoplasmacytic dermatitis and amyloidosis .
These c auses s hould be ruled out before a diagnosis of multiple myeloma is made
in a patient with an IgG monoclonal gammopa thy.

 A:G Ratio

 This is the ratio of albumin present in serum in relation to the amount of globulin.
The ratio can be interpreted only in light of the total protein c oncentration. Very
generally speaking, the normal ratio in most species approx imates 1:1.

For example, high total protein with a normal A:G ratio suggests dehydration,
while the same protein with a low A:G ratio would indicate hyperglobulinemia
 Blood Glucos e
 Glucose is derived from digestion of dietary carbohydrates, breakdown of
glycogen in the liv er (glycogenolysis) and production of glucose from amino ac id
prec urs ors in the liv er (gluc oneogenes is). In ruminants, the main source of
glucose is gluc oneogenesis from v olatile fatty acids (prioponate) absorbed from
rumen by bacterial fermentation. Glucose is the principal sourc e of energy for
ma mmalian c ells. Uptake is mediated by a group of me mbrane transport proteins,
called glucose transporters (GLU), s ome of whic h are insulin-dependent, e.g.
GLU-4.
 The blood gluc os e c onc entration is influenced by hormones whic h facilitate its
entry into or remov al from the circulation. The hormones affect gluc ose
concentrations by modifying glucos e uptake by cells (for energy production),
promoting or inhibiting gluc oneogenes is , or affecting glycogenesis (glyc ogen
production) and glyc ogenolys is. The most imp ortant hormone inv olv ed in gluc ose
metabolis m is ins ulin.
 Hormonal influences on blood gluc ose
 Ins ulin: Insulin enables energy use and storage and decreas es blood gluc os e
concentration. Insulin is produced by beta cells in the panc reatic is lets. Insulin
release is stimulated by gluc os e, amino acids and hormones (e.g. glucagon,
growth hormone, adrenaline). Release is inhibited by hypoglycemia, somatostatin,
and noradrenaline.
 Ins ulin decreases blood gluc ose by promoting glucose uptake through GLU-4 and
its use in metabolism (e.g. energy produc tion, protein production) by liv er, muscle
and other tiss ue cells. Ins ulin also inhibits glucose production by inhibiting
gluconeogenesis and glycogenolysis.
 Ins ulin increases fatty ac id and triglyc eride s ynthesis (through s timulation of
endothelial lipoprotein lipase), thus increas ing fat stores (adipogenesis), and
enhanc es glycogen synthes is and storage in the liv er.
 Ins ulin induc es the cellular uptake of K+, phosphate and Mg+.

 Hormonal influences on blood gluc ose contd.
 Sev eral hormones oppose the ac tion of insulin and, therefore, will increase blood
glucose. The main hormones that mediate this effec t are glucagon, growth
hormone, c atecholamines, and cortic osteroids. The increase in blood gluc os e c an
occ ur through inhibition of ins ulin releas e, stimulation of gluc ose-yielding
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pathways (glyc ogenolys is , gluconeogenes is), or decreas e of glucose uptake or

use by tissues . Collec tiv ely, increases in these hormones can induce a s tate of
ins ulin resistance. Ins ulin resistance can also be mediated by inflammatory
cytokines (TNF-alpha), obes ity and pregnanc y. Inflammatory c ytokines are
thought to be respons ible for insulin resistance observ ed in sepsis. Pregnanc y-
ass oc iated hormones may also c ontribute to insulin res istance and hyperlipidemic
syndromes in pregnant horses, ponies and c amelids.
 Hormonal influences on blood gluc ose contd.
 Glucagon: Glucagon causes an increase in blood gluc os e, by s timulating
gluconeogenesis and glycogenolysis and facilitating gluc ose release from
hepatoc ytes. Low blood gluc ose is the main sti mulus for glucagon release from
alpha cells in pancreatic is lets.
 Catecholamines (epinephrine/norepinephrine): Epinephrine from the adrenal
medulla acts via beta-adrenergic receptors , whereas norepinpherine is released
from nerv e endings and acts on alpha2-adrenergic receptors. Norepinephrine and
epinephrine have somewhat opposing effec ts on ins ulin releas e (norepinephrine
inhibits, epinephrine stimulates ), but the net effect of both is inc reased blood
glucose. This occurs v ia stimulation of glycogenolysis and releas e of gluc ose from
hepatoc ytes (epinephrine), and indirec tly through inhibition of ins ulin release
(norepinephrine), and releas e of growth hormone (epinephrine) and ACTH (which
inc reases cortis ol). The increase in gluc os e in response to catecholamines is
usually trans ient (primarily due to intermittent releas e of c atec holamines) and can
be quite pronounc ed in cats , c attle and cameli ds.

 Hormonal influences on blood gluc ose contd.
 Growth hormone (GH): This increases blood glucose by inhibiting gluc ose uptake
by cells . It als o promotes glycogenolysis in muscle tissue. Progesterone may
cause insulin resistance by stimulating secretion of GH. Growth hormone is
released from the pituitary by growth hormone-releasing hormone, whic h is
secreted by the hypothalamus usually in response to low blood gluc ose and
epinephrine.
 Corticos teroids : These inc rease blood gluc ose by inducing gluc os e releas e from
hepatoc ytes and inhibiting gluc ose uptake by c ells (through decreas ing GLU-4).
Corticos teroids also s timulate gluconeogenesis and gluc agon s ecretion (which
als o increas es blood glucos e).


 The table below summarizes the effects of thes e different major hormones on
physiologic processes that affect blood gluc ose conc entrations

 Sample considerations
 Serum - s erum glucose values decreas e rapidly in s amples that hav e not been
separated from the cellular c onstituents of blood. Gluc os e v alues decrease by
10% per hour if s erum is left in c ontact with cells . Note that the decrease in
glucose is enhanc ed in patients with inc reased leukocyte or platelet c ounts, ev en
if c ollec ted into fluoride oxalate tubes.
 Sodium fluoride (NaF) at c onc entrations of 10 mg/dl blood will inhibit gluc olysis by
erythroc ytes, leukoc ytes and platelets . Howev er, sodium fluoride is hypertonic and
causes lys is of red blood cells. This releases intra-erythrocyte water which dilutes
the glucos e concentration. Gluc ose conc entrations in sodium fluoride s amples are
consequently lower than in promptly separated serum samples (by approximately
7-12%).
 Lipemia and hemolysis may interfere with methodology

 Hypoglycemia
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 Lac k of gluc ose produc es seizures as the brain relies entirely on glucos e for its
energy source. Neonatal animals are predis pos ed to hypoglycemia due to
immature hepatic gluconeogenic pathways, low fat stores and musc le mass and
rapid glyc ogen depletion. Hypoglycemia can be due to dec reased produc tion (e.g.
inherited disorders, liver disease) or increas ed use (e.g. insulinomas, s epsis).
 Hypoglycemia contd.
 Artifact: Improper sample handling - e.g., mailing s erum on clot; high doses of
vitamin C c an inhibit some assays used to meas ure glucos e.
 Decreased production
1) Inherited defects : Hypoglycemia is a feature of certain glycogen storage
dis eases , namely deficienc ies of alpha 1-4 gluc os idase (Pompe dis ease) and
glucose-6-phosphatase (von Gierke's diseas e).
2) Idiopathic: Juv enile hypoglycemia (usually affects small breed dogs ).
3) Liver disease: Sev ere liv er disease and portos ys temic shunts c an produce
hypoglyc emia. More than 70% of the functional liv er mass must be los t before
hypoglyc emia ens ues .
4) Dec reased intake: Starvation, malabsorption. In horses, gluc ose decreas es if
they are fed a high grain diet, with little roughage.

 Hypoglycemia contd.
 Inc reas ed utilization
1) Idiopathic: Hypoglycemia of hunting dogs and endurance hors es .
2) Neoplasia: Insulin-sec reting tumors (insulinoma) or tumors secreting insulin-like
growth fac tors (mesenchymal tumors, hepatic tumors).
3) Sepsis: Hypoglycemia oc curs due to liver dysfunction, impairment of insulin
degradation and enhanced gluc ose utilization.
 Hypoglycemia contd.
 4) Bov ine ketos is , ovine pregnanc y toxemia: During pregnanc y, there are
inc reased glucose demands from the fetus and the mammary glands (plasma
glucose is the source of lactose). Ruminants are predispos ed to hypoglycemia in
pregnancy or lactation as they rely on gluconeogenes is for gluc os e produc tion.
Bov ine ketosis results in anorexia, depress ion, decreased mild production,
ketone mia, ketolac tia, ketonuria and hypoglycemia. It usually occurs in dairy cows
in the first 1-2 months of lac tation due to inc reas ed demands for gluc ose by the
ma mmary gland. It is initiated by poor diets or inappetance from other dis eases .
Alimentary ketosis results from feeding c attle s poiled silage with excess butyric
acid. A spontaneous ketosis als o occurs in c ows in peak lactation despite
abundant, good-quality feed. The animls are not ac idotic and often rec over
spontaneously (despite decreased milk produc tion). Beef c ows can also dev elop
ketosis, espec ially in the las t 2 months of pregnanc y, when carrying twins.
Ov ine pregnancy toxemia oc curs in sheep, carrying more than 1 fetus, that are
calorically deprived or stressed. They, like beef cows , develop fatty liv er and
acidos is and may die of liv er dysfunc tion.
Ketos is has also been reported rarely in lactating dairy goats and dogs.

5) Addis on's dis eas e: Hypoglycemia occurs due to decreased gluc oneogenes is
and increased insulin-mediated glucos e uptake by s keletal mus cle.


Hyperglyce mia

 Sustained hyperglyc emia c aus es glycosylation of protein groups. The firs t c hange
is the nonenzymatic addition of glucos e to protein amino groups to form Amadori
products. These reach a s teady state over time and do not accumulate further.
Any cause of hyperglyc emia (trans ient or s ustained) may result in glucosuria if
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glucose concentrations are high enough to exc eed the renal thres hold. The renal
threshold for gluc ose is spec ies -dependent and is reported to be 180-220 mg/dL
in dogs, 280-290 mg/dL in c ats (lower thresholds may occ ur in diabetic c ats ), and
150 mg/dL in hors es and cattle.
 Physiologic: Physiologic hyperglycemia occurs post-prandially and in respons e to
stress in all species . This can be mediated by epinephrine (and is transient, lasting
4-6 hours) or corticosteroids (results in a more sus tained increase in glucos e).
Cats and cattle tend to produc e marked s tress hyperglyc emias . In cattle, a v ery
high gluc ose (> 500 mg/dL) is a poor prognostic indicator. In liver dis ease, a
prolonged pos tprandial hyperglycemia may be obs erved.
 Therapeutic agents : Glucocorticoids, dextrose-containing fluids, thyroxine,
xylazine, megesterol acetate etc .

 Hyperglyce mia contd.
 Disease
Sustained inc reas es in glucose c an be seen with insulin deficienc y (type II
diabetes mellitus) or insulin resistanc e. Insulin res istanc e can be a res ult of
inc reased concentrations of hormones (e.g. gluc ocortic oids, growth hormone,
progesterone) or inflammatory cytokines (TNF-a) that oppos e ins ulin releas e or
the action of ins ulin on peripheral tissues. Obes ity is also ass oc iated with insulin
resistanc e, partic ularly in cats and likely in horses . Adipose tissue is now known to
be an endocrine organ and can produce specific hormones (e.g. leptin) as well as
inflammatory cytokines (TNF-a).

1) Diabetes mellitus: This is inherited in Keeshonds and Golden Retriev ers . It has
been associated with BVD infection in cattle and paramyxov irus infection in
lla mas. Cats are prone to non-insulin dependent diabetes mellitus . This is thought
to be associated with depos ition of pancreatic amyloid (from a mylin protein), whic h
is related to pancreatic islet dys function. When is let des truction is wides pread,
cats do become ins ulin-dependent.

 Hyperglyce mia contd.

 2) Hyperadrenocortic ism: In dogs and hors es, hyperglycemia is due to ins ulin
resistanc e.
3) Acromegaly: Hyperglyce mia is due to insulin resistance.
4) Hypergluc agonemia: Hyperglycemia is due to insulin resistanc e.
All the above produce prolonged or s ustained hyperglyc emia.
5) Hyperthyroidis m in cats : Increases in gluc os e are often trans ient. The exac t
mechanis m is unknown (? defec tiv e insulin secretion, ? enhanced sensitiv ity to
catecholamines ).
6) Acute pancreatitis: Hyperglycemia, which is usually trans ient, may oc cur due to
stress , glucagon secretion and decreased insulin production.
 Hepatocellular Leakage Enzymes

 The hepatoc ellular leakage enzymes are us eful in detecting injury to liv er
parenchymal cells . Generally, increas ed serum activity repres ents enzyme
leakage from cells through damaged cell membranes.
 AST: Us ed in s mall and large animals . Pres ent liv er as well as s keletal mus cle
and erythroc ytes.
 ALT: Us ed in s mall ani mals only. Largely liv er-spec ific, but can als o increas e in
sev ere myopathies (release of muscle enzyme) and hemolysis .
 SDH: Liv er specific in nearly all s pecies. Used in large animals in place of ALT
(whic h is not a good marker of liv er disease in large animals).
 GLDH: Liv er s pec ific in all species. Used in large animal panels concurrently with
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SDH (due to s uperior storage stability) and on exotic (non-mammalian) panels as

a marker of liv er injury.

 Disease effects
 Serum ac tiv ity of the leakage enzymes increas es when a s ufficient number of
hepatoc ytes experienc e increased membrane permeability. Serum lev els depend
on both the number of c ells affected and the sev erity of injury to indiv idual c ells.
Serum lev els do not c orrelate with reversibility. Diffuse hypox ia (reversible injury)
ma y result in greater serum activity than end-stage cirrhosis (irrev ersible injury).
Inc reas es are not specific with regard to the nature of the injury

 "Primary" hepatic disorders
 inflammation: Viral, bacterial, fungal, immune-mediated, idiopathic. Infla mmation
can be suppurativ e or non-suppurative. Note that primary bile duc t obstruction
ma y result in secondary hepatocellular injury as accumulated bile acids are toxic
to cells. Leakage enzyme lev els in end-stage liv er dis ease or portos ys temic
shunts may be normal or only mildly increased due to reduced number of cells
and minimal active injury.
 intoxications: Drugs, chemicals, plants . These can c aus e v ery high enzyme
activity if ass ociated with diffus e necrosis.
 neoplasms: Hepatocellular and bile duct carc inomas, metastatic neoplas ia.
Variable increases are poss ible depending on the ex tent of ac tiv e hepatocellular
injury.


 Disorders with secondary hepatic effects
 circ ulatory: Heart failure, shock, severe anemia, portosystemic s hunts, septicemia,
gastrointestinal dis ease in horses (displaced colon, ac ute colitis).
metabolic: Endocrine disease (produc ing fatty liv er, e.g. diabetes mellitus, Cushing's
disease, and idiopathic lipidosis), hyperthyroidis m, acute panc reatitis.
 Aspartate aminotransferase - AST
Glutamic ox aloac etic transaminas e, GOT

Aspartate amino transferas e c atalyzes the transfer of the alpha amino group of
aspartic acid to alpha-ketoglutaric acid, resulting in the formation of ox aloac etic
acid and glutamic acid. AST is useful as an indicator of liver and/or muscle injury
in large and s mall animals.

 Caus es of increas ed AST

 Artifact: Hemolysis or leakage from c ells c an cause erroneously high v alues
(enzyme is present in RBC).
 Drug effec ts: Anticonvulsants may cause an increas e in AST, whic h is thought to
be sec ondary to hepatocellular injury in dogs. Corticos teroids generally do not
result in increas ed AST levels, unless they result in a steroid hepatopathy (in
dogs).
 Physiologic effects : In horses , ex erc ise c an inc reas e s erum ac tiv ity as much as
30%. In early training, res ting levels are 50-100% greater than resting lev els of
hors es not in training.
 Disease effects

1) Myopathies : Muscle trauma (inc luding "down" animals), rhabdomyolysis, white

muscle diseas e (v itamin E-selenium defic iency), and infec tious myositis (black leg
or Clostridial myositis), and musc ular dystrophy may result in marked increas es.
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Serum CK ac tiv ity wil l also inc reased. Note that as AST has a longer half life than
CK, increases in AST persist for longer than increas es in CK. Therefore, in chronic
muscle diseas e, AST may be elev ated, whilst CK lev els may be normal. When
there is ac tiv e mus cle disease, both CK and AST are elevated (and CK will
dec line more rapidly as the injury res olv es

 2) Liver disease: AST will increase in liv er disease with the s ame caus es as for
ALT. Inc reas ed lev els seen with hepatocellular injury often aren't as high as those
seen with musc le damage. CK lev els are normal unless there is concomitant
muscle diseas e. Other liver specific enzymes (SDH) would also be inc reased. In
cats, AST appears to be a more sensitive marker of liv er injury (v alues are often
mildly increased with normal ALT in conditions s uch as pyogranulomatous
hepatitis sec ondary to feline infectious peritonitis v irus infec tion).

 Alanine aminotrans ferase - ALT
 Alanine amino trans ferase catalyzes the trans fer of the alpha amino group of
alanine to alpha- ketoglutaric acid, resulting in the formation of pyruvic and
glutamic acid.
Serum half-life is 59 hours in dogs and < 24 hours in c ats . Following ac ute hepatic
injury, serum enzyme activ ity peaks at about 48 hours and then begins to
decreas e.
Inc reas es in the enzyme occur due to cell damage (increased membrane
permeability or necrosis ) and induction (inc reas ed synthesis).

Organ s pecificity

ALT is v irtually liv er spec ific in dogs, cats, rabbits , rats and primates . Some
inc reases are possible in severe musc le dis eas es of the dog and cat due to
release of enzyme from this tissue. ALT is found in the liv er, mus cle (c ardiac and
skeletal), kidneys , and erythocytes .

Caus es of increas ed ALT

 Artifact: Hemolysis will caus e increased levels in the cat. Cats have a high RBC to
plasma ALT ratio. Hemol ys is has a minimal effec t on ALT in cattle, hors es , and
dogs.
 Drugs : Anticonvuls ants (primidone, phenobarbitone, dilantin) inc reas e ALT 4 x
normal. Although these drugs may induce ALT s ynthes is , increases in ALT are
thought to be s econdary to hepatoc ellular necros is.

 Disease effects
1) Liver disease: Both primary and s econdary hepatic disease can c aus e
inc reased ALT lev els , if altered cell membrane permeability or nec ros is occur.
Usually ALT v alues exceed AST v alues in liver disease. Hepatic neoplasia c an
result in marked inc reases in ALT in dogs , although increas es in AST are often
higher than the increases in ALT. Bile duct obs truc tion will increase ALT (and
AST) due to the tox ic effects of retained bile salts on hepatoc ytes. Trauma will
often increase ALT lev els, even without morphologic evidence of c ell injury.

2) Musc le disease: In large animals , ALT will increase with mus cle injury but is not
more us eful than AST in this regard so it is not included on large animal chemistry
panels . In small animals with sev ere musc le injury (isc hemic myopathy in cats,
muscular dystrophy in dogs), ALT will increase with CK and AST. Ho wev er, the
inc reases in ALT are usually less than increases in AST in primary musc le
dis ease and SDH v alues should be normal (unless there is conc urrent liver injury).

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 Sorbitol dehydrogenas e - SDH

Iditol dehydrogenase (ID)

Sorbitol dehydrogenas e is found in highes t c onc entration in the liver. It is a
cytoplas mic enzyme with a s hort half life (<4 hours in the dog). It c atalyzes the
conversion of fructose to sorbitol. It is a very spec ific indicator of liver dis eas e in all
species, although inc reas es can occur with primary or s ec ondary liv er dis eas e
(e.g. many hors es with inflammatory gas trointes tinal dis eas e will hav e high SDH
values). Increases occ ur within 24 hours of liver injury. SDH is the enzyme of
choic e for detecting hepatocellular injury in horse and cattle

SDH is not a stable enzyme. In cattle, serum samples are only s table for 5 hours
at room temperature, 24 hours at 4 C and several days frozen. In hors es , it has
similar s tability.

 Serum Alkaline Phos phatas e
Gamma Glutamyl Trans peptidase

 The primary utility of these two enzymes is to serve as indicators of cholestasis.

Cholestasis implies impairment of bile flow, whic h can be caus ed by physical or
functional obstruction of the biliary tract within or outside of the liver. When s evere
enough, cholestasis will result in elevations of bilirubin in blood. The main v alue of
these enzymes is their greater sensitiv ity for this abnormality as compared to
serum bilirubin lev els alone.

Alkaline phos phatas e, howev er, is less than entirely s pec ific for this purpose, in
that its ac tiv ity in serum can be influenced by a variety of other factors.

Gamma GT is more s pecific in general, and more s ens itive in some ins tanc es .

Alkaline phos phatas e (AP, ALP, SAP)

Alkaline phos phatas e is a non-specific metalloenzyme which hydrolyzes many

types of phos phate esters at an alkaline pH in the presence of zinc and
magnesium ions . There are different isoenzymes (gene products) and isoforms
(pos ttranslationally modified gene products ). The main use of ALP is as a
sensitiv e indicator of cholestas is in the dog (it will inc rease before bilirubin),
however it is non-s pec ific because c ortic osteroids (ex ogenous or endogenous
"stress ") induce increases in this enzyme. In the c at, ALP is a very spec ific
indicator of liver dis ease, whereas in large animals , the enzyme is not very useful
as it is insens itive, cholestatic dis orders are infrequent, and reference interv als are
quite broad.

Organ specific ity

 Liv er - hepatoc ytes, epithelium of biliary tract. Thes e c ells are the s ource of the L-
ALP isoform (all species) and C-ALP isoform in the dog.
 Bone - this isoform is produc ed by osteoblasts and increases in serum in
ass oc iation with osteoblas tic ac tiv ity (young animals , certain bone dis orders).
 Intestinal, renal, mammary, placental tissues - Leukocytes - ALP is found within
myeloid cells, inc luding neutrophils, eos inophils and monocytes . Cell lineage
expression is species-dependent, i.e. monoblasts (immature monocytes) in dogs
are particularly rich in ALP.

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 Leukocytes - ALP is found within myeloid c ells, inc luding neutrophils, eos inophils
and monoc ytes. Cell lineage ex press ion is species-dependent, i.e. monoblas ts
(immature monocytes) in dogs are particularly rich in ALP.

 Drug effec ts
 Glucocortic oids: In dogs, increased total ALP is due mainly to s ynthesis of the C-
ALP isoform. Marked increases are poss ible (50-100 fold). Total ALP may remain
high for three to six weeks , depending on the drug preparation administered (ie,
short-acting v s. depot forms ). Interes tingly, it takes approx imatel y 10 days for C-
ALP to be induced by c orticosteroids; therefore the initial increases in total ALP
with corticosteroid administration is due to increases in the L-ALP, and not the C-
ALP, isoform.
 Anticonvulsants : phenobarbital, primidone, phenytoin - mild to marked increases
in total ac tiv ity occur, due mainly to raised L-ALP is oform. This is probably
sec ondary to choles tasis because studies in dogs with phenobarbitone show that
liv er s ynthes is of ALP is not induced.
 Age effects
ALP activity in young, growing animals of all species may be 2 - 10 times higher
than in adults , due to increased B-ALP isoform. Values decrease within 3 months
of age and are within adult ranges by 15 months of age.

 Disease effects
 Hepatobiliary diseas e: Increases in ALP (primarily the L-ALP isoform) is us ed as
an indicator of cholestasis (intra- or ex trahepatic ) in animals. In c ats , ALP is a
specific but ins ensitive marker of hepatobiliary dis eas e. Increas es in ALP do occur
in hepatobiliary dis ease, but the inc reas e is less reliable and of lower magnitude
compared to the s ituation in dogs (feline hepatic tiss ue c ontains muc h less ALP
and serum half life is only six hours ). Therefore, any increases in ALP in the cat
are considered clinically relevant. The wide range of ALP activities and
ins ens itivity of this tes t to cholestas is in large animals li mits utility of ALP in these
species.
a) Ex trahepatic choles tasis (bile duc t obstruction): This c aus es very dramatic
inc reases in ALP. Increas es in ALP may occ ur before development of icterus ,
espec ially in the dog. Pancreatitis (acute or chronic ) may result in increas ed ALP
lev els from s welling and/or fibrosis around the bile duc t (es pec ially in cats which
hav e a common bile and panc reatic duc t).

 b) Intrahepatic choles tas is : Localized or generalized cholestasis from hepatoc yte
swelling will induce ALP. Lesions that are primarily centrilobular generally cause
only mild increases in ALP while lesions affecting the periphery (periportal areas)
of the lobule usually result in more dramatic elev ations as a res ult of impaired bile
flow. Caus es of intrahepatic cholestasis include neoplasia (primary or metas tatic),
hepatic lipidos is (marked inc reases are poss ible with idiopathic hepatic lipidosis in
cats - lipidos is is the caus e of the most dramatic increases in ALP in this s pec ies ,
often without concurrent elevations in GGT, which is a useful diagnos tic feature),
acute hepatoc ellular injury (intrahepatic choles tas is occurs due to hepatocellular
swelling; elev ated ALT lev els are ex pected c onc urrently), bile sludging (occ urs
with anorexia, especially in cats ) and periportal fibrosis and inflammation (marked
inc reases are possible).


 c) Func tional cholestasis : This is defined as decreased bile flow due to
downregulation or inhibition of transporters res ponsible for exc reting bile s alts or
conjugated bilirubin into bile. It occ urs without any physical obstruction or
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impairment to bile flow. It is frequently mediated by inflammatory cytokines and

has been reported in dogs with E coli infections . It likel y occ urs in other species as
well. Cyto kine-mediated cholestasis is usually c haracterized by high total bilirubin
(due to direc t and indirec t bilirubin) with mild i ncreas es in hepatoc ellular leakage
enzymes (ALT, SDH, GLDH). ALP lev els may be normal in this condition.
d) Neoplasia: In primary liv er cancer (hepatocellular/biliary), marked increases in
ALP are poss ible (due to L-ALP or C-ALP in dogs). Metastatic neoplasia often
inc reases ALP due to localized cholestasis . In many cases of hepatic neoplas ia,
ALP may be the only enzyme that is increased on a panel.

 e) Acute hepatoc ellular injury: Mild to moderate elev ations in ALP are attributed to
intrahepatic c holes tas is assoc iated with hepatoc ellular swelling rather than
hepatoc ellular injury per se. Concomitantly high values of ALT, SDH and GLDH
would be ex pec ted.
 Hyperadrenoc orticis m - Levels v ary from moderate to marked (up to 100- fold) and
are frequently due to induction of the C-ALP isoform (although L-ALP increas es
are als o s een) in dogs. Up to 83-100% of dogs with Cushings dis eas e have high
C-ALP lev els, but chronic endogenous stress (due to any underlying dis eas e) may
inc rease C-ALP and total serum ALP (up to 2-3 x normal). Therefore, C-ALP
lev els are a sensitive, but not spec ific, test for hyperadrenocorticism in dogs.

 Inc reas ed osteoblas tic ac tiv ity - osteoblastic activity in response to hormones
(PTH, thyrox ine) or neoplasia (osteos arc oma) may inc rease total serum ALP due
to the B-ALP is oform.
1) Primary and s econdary hyperparathyroidism (2-3x inc rease).
2) Osteos arcoma
3) Fracture healing in dogs .
4) Hyperthyroidis m in cats : Affected animals may hav e mild increases in total
serum ALP, which is mostly due to high B-ALP with a lesser inc reas e in the L-ALP
isoform.

 Gamma-glutamyl transferas e - GGT

Gamma-glutamyl transpeptidase, GGTP

Organ s pecificity

GGT is found primarily in the membrane and in mic ros omes (from SER) as
aggregates . A s mall portion (< 5%) is found in the c ytoplasm. Dis aggregation
(solubilization) and increased synthes is result in increas ed activity in s erum.
 Biliary trac t - sourc e of serum ac tiv ity in health and dis ease.
 Panc reas, gastrointestinal tract - GGT does not increase in serum in dis orders
inv olv ing these tissues (it is usually s hed into the lumen of these organs ).
 Kidney - prox imal renal tubules. GGT is shed into urine, rather than blood.
 Mammary glands - GGT is excreted into milk.
 Reproduc tiv e tract - Epididymis (GGT is secreted into semen).



 Caus es of increas ed GGT
 Drug effec ts
Inc reas es in GGT occ ur s econdary to therapeutic drugs caus ing cholestasis.
Inc reas es may also be seen with antic onvulsant or cortic osteroid therapy,
pres umably s ec ondary to liver injury caus ing loc alized c holes tas is or biliary
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hyperplas ia. Induction of GGT synthes is may also contribute to high GGT lev els
with corticosteroids. Increas es in GGT occur as soon as 5 days after c orticos teroid
administration.

 Physiologic
a) Neonates: Colos trum in all s pec ies , except for horses. contains high GGT
concentrations. Increas es in GGT occur within 24 hours of suc kling and are a
sensitiv e indicator of passive transfer.
 b ) Breed: Donkeys and burros have 2-3 x GGT lev els of horses .


 Disease effects
Inc reas es in GGT occ ur s econdary to hyperplasia or induction of s ynthes is .

a) Hepatobiliary dis ease: In s mall ani mals, GGT is a sensitiv e indicator of biliary
hyperplas ia and cholestas is .
 b) Renal dis eas e: GGT is ex press ed on the membranes of proximal renal tubular
epithelial cells. Cell injury c aus es GGT to be shed into the urine and not into
blood. Urinary GGT:creatinine ratio has been s tudied as an early indic ator of renal
tubular injury.

 Bilirubin
 The bilirubin panel provides information about the total bilirubin c oncentration in
the sample, as well as the c ompos ition of that total in terms of proportion
conjugated vs. unc onjugated
 Total Bilirubin
 The majority of bilirubin (80%) is produc ed from the degradation of hemoglobin
from erythrocytes undergoing normal (remov al of aged or effete cells ) or abnormal
destruc tion (i.e. intrav ascular or ex trav ascular hemolysis) within mononuclear
phagoc ytes (princ ipally s plenic , hepatic and bone marrow macrophages). A small
perc entage (20%) is derived from the catabolis m of v arious hepatic hemoproteins
(myoglobin, c ytochrome P450) as well as from the ov erproduction of heme from
ineffec tiv e erythropoies is in the bone marrow. W ithin mac rophages, a free heme
group (iron + porphyrin ring) is oxidized by mi cros omal heme oxygenase into
biliv erdin and the iron is released (the iron is then s tored as ferritin or released into
plasma, where it is bound to the trans port protein, transferrin).

 ). Biliv erdin reductase then reduc es the green water-soluble biliverdin into
unconjugated bilirubin. Heme ox ygenase is als o located in renal and hepatic
parenchyma, enabling thes e tiss ues to take up heme and convert it to bilirubin.
Birds lack biliv erdin reduc tas e, thus they excrete heme breakdown produc ts as
biliv erdin rather than bilirubin. Unconjugated or free bilirubin is then releas ed into
plasma where it binds to albumin. Uptake of unc onjugated bilirubin occurs in the
liv er and is c arrier-mediated. The carrier-mediated uptake is shared with
unconjugated bile acids and dyes s uc h as BSP. Once within the hepatocyte,
unconjugated bilirubin is transported with ligand (Y protein) or other proteins (e.g.
Z protein) and the majority is conjugated to glucuronic acid by UDP-glucuronyl
transferase. The remainder is conjugated to a v ariety of neutral glyc os ides
(glucose, xylose). In the horse, the majority of bilirubin is c onjugated to gluc os e.
Bilirubin mus t be conjugated before it can be excreted into bile (c onjugation
ma kes bilirubin water soluble). Excretion into biliary canaliculi is the rate-limiting
step of the entire bilirubin metabolism pathway and occ urs via spec ific
transporters , which are energy (ATP) dependent

 Trans fer into the canaliculi is fac ilitated by bile salt-dependent and bile salt-
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independent biliary flow (the latter of whic h is generated by a basolateral

(sinus oidal or blood-side) Na/K ATPas e pump). Bilirubin is sec reted, along with
bile s alts (and s odium) into the intestine, where the bile salts form micelles
fac ilitating abs orption of fat. In the intes tine, bacterial enzymes deconjugate
bilirubin and degrade it to urobilinogen. Urobilinogen is reabs orbed (about 10%) or
broken down (90%) into urobilin and stercobilin (both of which are excreted in the
feces). Of the res orbed urobilinogen, most is taken up by the liv er (enterohepatic
circ ulation, i.e. the urobilinogen is absorbed into the portal v ein, taken up by the
liv er and re-excreted into bile), whils t the res t bypass es the liver and is exc reted
into the urine. Conjugated bilirubin is not normally found in the urine of domes tic
animals, although s mall to 1+ amounts of c onjugated bilirubin may be seen in
concentrated urine from dogs (particularly mal es), due to the low c anine renal
threshold for bilirubin. In all spec ies (but dogs, in particular), bilirubinuria may
prec ede an inc rease in serum bilirubin in cholestatic disorders . Remember, only
conjugated bilirubin can be excreted in urine as it is water soluble.

 Circulating bilirubin exists in two main forms as determined by the Van den Bergh
reac tion, which differentiates bilirubin into conjugated (direc t) and unc onjugated
(indirec t) forms . There is a third form of bilirubin, called delta bilirubin (or
biliprotein), which is conjugated bilirubin bound to proteins . Delta bilirubin
inc reases in serum when hepatic excretion of conjugated bilirubin is impaired
(cholestas is ) and the liver retains intac t conjugation mechanis ms . Delta bilirubin
ma y be res ponsible for a persis tent bilirubinemia without bilirubinuria seen in
some animals with c holestasis.
 Caus es of increas ed total bilirubin
 Artifact: W ith some analyzers and reagents, hemol ys is and lipemia (ev en mild) will
cause artifac tually high bilirubin v alues.
 Hemolysis : Des truction of red cells , whether through extravascular or intravascular
hemol ys is will inc rease the production of unconjugated bilirubin because of
enhanc ed hemoglobin metabolism by mononuc lear phagocytes .
 Liv er diseas e: Hepatic diseas e may cause increases in both unconjugated and
conjugated bilirubin. Increases in bilirubin in dogs often occur after elevation of
cholestatic enzymes (GGT, SAP) due to the low renal threshold for bilirubin. In
acutely dev eloping ic terus, SAP and GGT levels may be normal because they
require time for induction. In large animals with liv er dis eas e, inc reas es in bilirubin
are usually due to unconjugated bilirubin.

 Cholestasis: This is defined as decreas ed bile flow and can be due to phys ical
obstruc tion of bile flow or functional defec ts in the transporters that deliver bile
salts or bilirubin into the biliary system. Phys ical obs truc tions to bile flow c an be
intrahepatic ((e.g. hepatoc yte swelling due to hepatic lipidosis in cats ) or
extrahepatic (e.g. bile duct obs truc tion from pancreatic neoplasia, cholelithiasis,
Fasciola hepatica in c attle). Func tional defects in bile salt or bilirubin trans porters
occ ur sec ondary to inflammatory cytokines (e.g. endotox emia) and drugs .
 Amylas e and Lipase
 Serum ac tiv ity of thes e enzymes is measured as an (imperfect) aid to the clinical
diagnosis of pancreatic injury.

Idiopathic inflammatory disease (acute-to-chronic panc reatitis) is the mos t

common diseas e entity (mainly dogs, occ as ionally c ats , rarely, horses ). Less
commonly, panc reatic tumors (adenoc arc inoma) or trauma (HBC) can be the
cause of c linic al signs and elevated enzyme activity.

With regard to the diagnosis of panc reatitis, res ults mus t be interpreted in light of
the his tory and c linical s igns in the patient, and correlated with physic al,
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radiographic, hematologic, and other c linical chemis try findings:


 vomiting, diarrhea, history of recent fatty meal, table scraps, garbage ingestion
 painful abdomen, dehydration, someti mes ic terus
 hazy cranial abdomen on plain films
 neutrophilic leukoc ytosis
 azotemia (often), lipemia (sometimes), hypocalcemia (occas ionally), cholestas is
 Newer tests , s uch as trypsin-like immunoreactiv ity (TLI) c an be used to diagnose
pancreatitis and are thought to be more sensitiv e than meas urement of these
enzymes in dogs.
 Amylas e
 Organ s pecificity
 Panc reas: Found in zymogen granules. The pancreas has higher concentrations
of amylas e than other tissues .
 Intestine: Duodenum, ileu m
 Ov ary and testes
 Saliv ary gland: Saliv ary amylase is found in high concentration in pigs, resultilng in
high reference intervals for amylas e in this species. Dogs lack saliv ary amylase.
 Caus es of hyperamylasemia
 Acute panc reatitis : The increase and decrease of serum a mylase tends to parallel
that of lipase. Amylase v alues peak at 12-48 hours and are normal within 8-14
days after about of pancreatitis in dogs. It is rare to observe increased amylas e in
cats with pancreatitis . Note that amylas e concentrations are often higher in ascitic
fluid than in blood in animals with pancreatitis (or intestinal dis ease).

 Chronic renal insuffic iency: As discuss ed abov e, the inc reas e in amylas e is due to
macroamylas e formation (polymeri zation with globulins ).
 Decreased GFR: This can c aus e increas ed amylas e (up to 2-3 x normal) in the
absence of s ignificant pancreatic diseas e. If an azotemic patient has amylas e
values greater than 2 to 3 times the referenc e v alues, pancreatitis should rec eive
consideration as a diagnos is .
 Intestinal dis ease/obs truc tion: Moderate elevations in amylas e are poss ible.

 Lipase
 Lipase hydrolyzes triglycerides . There are s everal forms of lipase: pancreatic
lipase, c olipas e and lipoprotein lipas e.

Caus es of increas ed lipase

 Drugs : Corticosteroids are reported to inc rease lipase values.
 Acute panc reatitis : Des truc tion of pancreatic ac inar tiss ue results in the esc ape of
pancreatic enzymes into the pancreas and peritoneal c av ity. The enzymes enter
the blood by wa y of lymphatics or c apillaries with s ubs equent elevation of serum
lev els . Increases of at least 2 x normal are seen in pancreatitis .
In the c at, lipase is not cons istently elev ated in panc reatitis . It was hoped that
measurement of tryps in-like immunoreactivity (TLI) would be better for diagnosis
in c ats, as high values are s een in dogs with pancreatitis and are very useful for
diagnosis in dogs with normal lipase values.

 Gastrointestinal disease: Peritonitis , gastritis , bowel obs truc tion, v isc eral
manipulation (laparotomy), hepatic dis ease and neoplasia may inc reas e lipase
values by 2-3 x normal.
 Decreased GFR: Increases of up to 4 x normal may be s een in patients with
decreas ed GFR. In azotemic patients with lipas e lev els greater than 3-4 times
normal, the diagnosis of pancreatitis should s till be considered.
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 Cholesterol
 Cholesterol is the most c ommonly oc curring steroid. It is an important precursor of
cholesterol esters, bile ac ids and s teroid hormones. It is derived from dietary
sources and synthesized in viv o from acetyl-CoA in the liver (main s ite) and other
tissues (intestines , adrenal glands and reproduc tiv e organs). The bile is the main
route of excretion of c holes terol. Note that v isible hyperlipemia in a blood sample
is usually due to increased triglyc erides not due to inc reas ed choles terol.

 Caus es of hypercholesterolemia

 High cholesterol is usually due to increased numbers of c holes terol-ric h

lipoproteins, i.e. HDL and LDL. Bec ause VLDL's do contain s ome c holes terol
(12%), high choles terol c an also be seen with v ery high VLDL conc entrations .
 Inherited disorders of lipid metabolis m: Familial hypercholesterolemia has been
reported in Briards, Rottweilers, Shetland Sheepdogs , and Dobermans.
 Diabetes mellitus : Insulin stimulates lipoprotein lipase, which is respons ible for
hydrolys is of c hylomic rons (CM) and VLDL. Insulin also antagonizes hormone
sensitiv e lipase, the hormone res ponsible for lipolysis of adipos e tissue. Lack of
inhibition of hormone sens itiv e lipase causes increas ed lipolysis, with increas ed
non-esterified fatty ac id pres entation to the liver and VLDL production. In addition,
LDL receptors on hepatocytes are downregulated, resulting in increased LDL
lev els .


 Hypothyroidis m: In dogs, hypothyroidism is assoc iated with mild to marked
elev ations in c holesterol, due to inc reased LDL and HDLs. Thyroid hormone (T3)
stimulates LDL rec eptors (and promotes uptake of c holes terol), therefore lack of
thyroid hormone results in decreas ed LDL receptors and decreased LDL
(cholesterol) uptake.
 Nephrotic syndrome: This is charac terized by edema, hypoalbuminemia,
hyperc holesterolemia and albuminuria and is c aused by glomerular damage, e.g.
amyloidosis, immune-complex glomerulonephritis. There is an inc rease in HDL
and LDL in this syndrome, although the exact mechanis m is unknown.
 Hyperadrenoc orticis m: Hypercholesterolemia is due to increased LDL, thought to
be due to peripheral insulin res istanc e and down-regulation of LDL receptors in
the liv er. Cortic osteroids als o s timulate hormone-sensitiv e lipase, resulting in
inc reased lipolysis and VLDL production.

 Cholestasis: In hepatobiliary disorders , especially thos e c aus ing ex trahepatic

cholestasis (e.g. bile duc t obstruction), inc reased cholesterol may be seen.
 Panc reatitis: Although hypertriglyceridemias are more common in this dis order,
high c holesterol may be s een concurrently due to inhibition of lipoprotein lipas e.
 Exc essiv e negativ e energy balance: In states of exc essive negativ e energy
balanc e (e.g. s tarvation, anorex ia) particularly when energy demands are high
(e.g. late pregnanc y, early lactation), lipolysis of fat stores in adipocytes will
inc rease VLDL c onc entrations . Although VLDLs contain more triglycerides than
cholesterol, inc reases in both of these analytes may be seen. Hyperlipemia due to
exc essive negative energy balanc e mostly occ urs in horses and c amelids and is
ass oc iated with sec ondary hepatic lipidos is.

 Caus es of abnormally lo w choles terol levels
 Low c holes terol can be due to decreased numbers of c holes terol-containing
lipoproteins (LDL, HDL, VLDL) or a decreas ed c holes terol content of these
 http://www.unaab.edu.ng

lipoproteins.
 Decreased absorption: Malabs orption and maldigestion problems, e.g. protein
los ing enteropathies , ex ocrine pancreatic insuffic iency.
 Decreased production: Sinc e the liver is the main site of cholesterol production,
low c holes terol values c an be seen in c hronic liv er diseas es (e.g. cirrhos is),
synthetic liver failure (acute or chronic), and portosystemic s hunts (ac quired or
congenital).
 Altered metabolism: Infla mmatory c ytokines can reduce the cholesterol content of
lipoproteins by dec reas ing lecithin-cholesterol acyltransferas e ac tiv ity (the enzyme
respons ible for c onv erting free choles terol to cholesterol ester which is then
inc orporated into HDL and LDL)..
 Inc reas ed uptake of lipoproteins: Upregulation of LDL-rec eptors on cells
(peripheral tiss ues and liv er) c an potentially lower c holesterol values. This occurs
in rapidly proliferating tumor cells (e.g. acute myeloid leuke mia in human patients)
and in respons e to inflammatory c yto kines (some acute phas e proteins in human
patients, s uch as s erum amyloid A, enhance LDL removal from the c irc ulation in
acute phase reactions).
 Creatine Kinas e – CK
 CK is a "leakage" enzyme present in high concentration in the cytoplas m of
myoc ytes and is the mos t widely us ed enzyme for ev aluation of neuromus cular
dis ease.
 Caus es of increas ed CK
 Artifact: Hemolysis will increase CK values as analytes in red blood cells (ADP or
G-6-P) or their membranes (adenylate kinase) contribute to the enzymatic reaction
for CK, artifactually inc reas ing values. Inadvertant penetration of muscle during
venipunc ture can caus e 3- to 4-fold increases in CK ac tiv ity in the sample and is a
common c ause of mildly inc reased CK values in healthy (or s ick) animals .
 Physiologic: CK values in young puppies are higher than in adults . Moderate
inc reases (2-3x) are poss ible in exerc is ing hors es . Pos t exerc is e increase is less
in well conditioned animals although the bas eline level is somewhat higher.
Intra muscular injections will increas e CK v alues (2-3x), dependent on the drug,

 mus cle binding of drug, loc al blood flow. CK wi ll especially increas e if the
injected compound is an irritant (e.g. tetrac yc lines).
 Muscle diseas e: Detection of inc reas ed activ ity in serum is useful as an indicator
of mus cle injury. The assay is quite s ens itive in this regard, but elevations are not
specific as to c ause (e.g., trauma, inflammatio n, degeneration). High CK v alues
are obs erved in inherited muscular dystrophies, exercise-induced rhabdomyol ys is,
polymyositis, vitamin E-s elenium deficienc y, snake bite poisoning, etc
