Lab 6 Metabolism Flashcards
Biuret method
what is it used for and what is the sensitivity
To check TP
CuSO4 containing
20-100g/l sensitivity
Average TP concentration of plasma
60-80 g/l
How can we measure TP lower than 20 g/l (urine, CSF, etc.)
Lowry method (Folin-phenol) Ultra sensitive TP method
Biuret test
Chemical reaction
colour of complex
wavelength
Photometrical measurement
purple colored complex
546 nm
Ultra sensitive TP analysis
Reagents
wavelength
sensitivity
Na-molibdate, pirogallol red
Binding of protein directly to stain
600 nm
0.2-4 g/l
Refractometry mechanism
errors that can give false results
calibration
sensitivity
light refraction characteristics of a solution depend on its specific gravity, the TP in blood has the biggest influence on the light refraction
specific gravity depends on temp (20)
hemolysis, lipaemia
distilled water
25-95 g/l
what happens to TP during hyper hydration or dehydration
Hyper: decreased TP
De: increased TP
Protein fraction of dog Plasma TP Serum albumin Serum globulin Albumin/globulin Fibrinogen
unit:
67-70
48-64% (25-34)
11-21% (30-48)
1.083
1-4
UNIT: g/l
Two methods to measure albumin
Spectrophotometry
Electrophoresis
Albumin spectrophotometry
Bromocresol green
binds to albumin pH 4.2
Blue-green complex 578 nm
Albumin serum electrophoresis
in combination with TP measurement
Protein fraction analysis
Albumin as a % of TP
Decrease of albumin:
intake
Decreased digestion or absorption
Decrease of albumin:
synthesis
liver failure, acute inflammation
albumin is a negative acute phase protein
Decrease of albumin:
utilization
physiological: pregnancy, work, exercise, production (egg,milk)
chronic diseases (infl, neoplasms)
Decrease of albumin:
loss
via kidneys: PLN via Gi tract: PLE via skin: burn whole blood loss hyperhydration
increase of albumin
dehydration
Globulin methods to measure
1: calculation: Tp - albumin ≈ globulin
2. serum electrophoresis: % (if TP is known)
Alb/Glob ratio
Decrease of ratio: inflammatory/neoplasia (inc globulin)
Inflammation can be analyzed by RBC sedimentation or glutaric-aldehyde test
Electrophoresis basic principles
Proteins have amphoteric character (can react both as acid and base)
serum is placed on gel and electric current is exposed to separate proteins into five categories based on size and electric charge
What influences rate of motion during electrophoresis
High charge and small size increases rate of motion
Low viscosity also increases speed
What is the efficiency of electrophoretic separation of ions determined by?
Relative charge densities (charge per unit volume)
Gel electrophoresis mediums
Polyacrylamide or agarose (minimizes diffusion)
SDS-PAGE
sodium docecyl sulfate polyacrylamide gel electrophoresis
Proteins denatured by heat
and coated by neg charged SDS
then the protein is applied to the cathode (-) part of the gel and the voltage in the gel forces the neg charged proteins to move toward the anode (+)
densitometer detects results after staining
alpha globulins
APP
beta globulins
IgA, IgM
LDL
gamma globulins
IgG
Monoclonal
One cell group of the same origin
Polyclonal gammopathy
ø diseases
increase of globulin conc
broad-based peak in beta/gamma region
bridging between beta and gamma globulins
polyclonal gammopathy
diseases
bridging
Chronic inflammatory disease Liver disease FIP (a2) (virus) Occult heartworm disease (parasite) Ehrlichiosis (bacteria)
bridging:
increased IgA IgM:
lymphoma, heartworm, chronic active hepatitis
Monoclonal gammopathy
Sharp spike in beta or gamma region
Monoclonal gammopathy neoplasia
5
Multiple myeloma (Plasma cell myeloma) G/A osteolytic bonelesions
Lymphoma M/G
Chronic lymphocytic leukemia G
Extramedullary plasmacytoma in skin of dogs
IgM increase: macroglobulinemia
B cell lymphoma, spleno-/hepatomegaly
monoclonal gammopathy
non-neoplastic
6
Rare usually IgG (should be ruled out) occult heartworm disease FIPV Ehrlichia vanja Lymphoplasmacytic enteritis/dermatitis Amyloidosis
Hypoglobulinaemia
Decreased intake: neonates (colostrum, malabs)
Decreased synthesis: inherited immunodeficiency, liver function impairment
Increased loss: PLN, PLE, skin, bleeding
Fibrinogen two methods
1: measurement of both plasma and serum TP (plasma - serum=fbg)
2: plasma TP is measured before and after heat 58 C, refractometry (fbg heat labile)
3: thrombin time
Fibrinogen increase
Acute inflammation (esp ru) Dehydration
Fibrinogen decrease
liver function impairment, protein deficiency (advanced) DIC Sequestration after body cav bleeding Chronic bleeding blood loss inherited afibrinogenaemia (st bernard)
Glucometer
Electric conductance (normocytaemia)
GOD/POD spectrophotometry
Glu + H2O = gluconic acid + H2O2 = O’ + H2O
O’ oxidizes stain from colorless (reduced form) to a colour that depends on glucose conc
In vitro catabolism of glucose
RbC catabolyzes glu: cooled sample plasma separated quickly coagulate RBC (3cl acetic acid) NaF in blood sample (inhibits enolase)
Transient glucose increase 6
Laboratory errors stress food intake xylazin effect cranial trauma or inflammation glucose containing fluid therapy
constant hyperglycaemia
4
Diabetes mellitus
Hyperadrenocorticism (cushings), GCS therapy
Progesterone
Enterotoxaemia of sheep (clostridium)
Decreased glu conc 11
Lab error (storage/transport) Decreased energy status (ketosis, baby pig, exercise) insulin overdose insulinoma anabolic steroids terminal liver failure acute liver failure hypoadrenocorticism (addisons) septicaemia hyperthyroidism paraneoplastic syndrome
IV glu tolerance test when is it done and what does it test
during suspicion of DM or insulinoma
2 starving measurements: >11 mmol/l
IV glu tolerance
how is it done
animal starved for 24hr blood sampling 0 min 1g/bw 40% glucose conc solution infusion 40 sec) blood sampling at 5, 15, 30, 45, 60 min normalization: 30-60 min
oral glu tolerance test (glu abs)
suspicion of chronic vowel disease, exocrine pancreatic insufficiency
blood sample 0 min
2g/bw 12.5% glucose solution given orally
blood sampling 15, 30, 60, 90, 120
twice normal: 30 min
normalized: 120 min
Macroamylase
glucose bound to alpha amylase
cannot be excreted by kidneys, circulated blood for a longer time
fructosamine
glucose bound to more than one protein (ketoamine)
conc not influenced by short term hyperglycemia
average blood glucose level 2-3 weeks before sampling
glycated hemoglobin
non-enzymatic glycation by hgb exposure to plasma glucose
average blood glu levels 2-3 months before measurement
Ketone bodies
Acetone, acetoacetate, beta-OH-butyrate
decreased intake of carbs or decreased insulin prod
Ross reaction
Acetone and acetoacetate conc
changes color from white to purple
depth depends on ketone conc
Relative conc of ketones in different body fluids
Milk: 1 mmol
Plasma: 3-5 mmol
Urine: 10 mmol
Urea of milk and plasma
Can determine energy status of dairy cattle
energy deficiency in rumen -> NH3 level increases -> urea production in liver increases
milk urea 3 -> 9
plasma urea 9 -> 16
What are chylomicrons in the blood a sign off?
post prandial (after eating)
How can you differentiate chylomicrons from other lipids in the blood plasma?
Freeze plasma -18, wait 12+hr and slowly warm and centrifuge. the protein part of the CM will coagulate, and leave a transparent layer under the fat. not transparent = lipid mobilization from fat stores
causes of hyperlipidaemia 9
ponies diet DM hypothyroidism hyperadrenocorticism/gcs therapy nephrotic syndrome septicaemia (energy def) pancreatitis idiopathic (schnauzers, beagles)
causes of decreased lipid content 3
long term starvation liver failure (eg PSS) malabsorption or maldigestion
Lipid absorption test
when is it done
To determine whether there is existing malabsorption, maldigestion (pancreatic insufficiency) or chronic bowel disease
What is normal plasma triglycerol (PT) and what is PT after fast lipid intake in normal conditions?
Normal (dogs) 1 mmol/l
Fast lipid intake 2 mmol/liter
how is the lipid absorption test performed?
24hr starvation
blood sample 0 min
3ml/bw corn oil po
blood sampling each hour
if no change: add pancreatic enzyme extract to corn oil
if change is seen: epi
if still no change: intestinal absorption defect
what is cholesterol measurements used for in vet practice?
detection of increased fat mobilization -> total cholesterol value increases
(hypothyroidism, hyperadrenocorticism, nephrotic syndrome, DM)
what is the fraction of cholesterol-ester of total cholesterol value?
40%
what can happen to cholesterol as a result of impaired liver function?
decreased esterificiation
and decreased apolipoprotein production (ldl, hdl, etc)
what is the normal value for cholesterol conc?
2-6 mmol/l
5 causes of hypocholesterolaemia
malnutrition liver failure (decreased synthesis) neoplastic disease hyperthyreosis (inc usage) decreased apolipoprotein synthesis
7 causes of hypercholesterolaemia
high dietary fat content hypothyroidism hyperadrenocorticism DM nephrotic syndrome (plus low TP) cholestatic diseases (bile duct obstruction) idiopathic
what can cause increased blood FFA conc?
high energy need (lactation)
how long can FFA compensate the energy deficiency?
until the liver can not produce any more OAC for beta oxidation
starvation, glycogen deficiency of liver, lipid mobilization syndrome and hepatic lipidosis leads to:
decreased TL (total lipid conc) because the liver can not produce enough apolipoproteins for transporting lipids
FFA conc is increased because they are transported by albumin
normal FFA value
0.1-0.3 mmol/l
TL conc
5-7 mmol/l
TG conc
sheep?
0.6-1.2 mmol/l
sheep has higher
1.5-4 mmol/l
cholesterol conc
2-6 mmol/l
VLDL
TAG
HDL
phospholipids
LDL/HDL
Cholesterol
(HDL)
cholesterol-ester
Albumin transports:
FFA
