hey folks welcome back to our lab review
sessions this one is going to talk about
clinical chemistry so I'm gonna quickly
go over review some of the major
biochemical markers that we look at in
fact medicine just so you have an idea
of what's what why we look at what we do
okay so we're gonna start talking about
protein assays so a couple of notes
about proteins so they're not
specifically a liver function indicator
but most plasma proteins are produced by
the liver so if we have a liver disease
or any change in liver function then
often we will get usually low plasma
proteins some proteins are produced by
the immune system so you'll remember
that one of the big proteins involved in
the immune system is immunoglobulin so
that's a globulin protein and so we
definitely get quite a few proteins from
their alterations of protein
concentrations occur in a variety of
disease conditions primarily liver and
kidney disease but also gastrointestinal
disease skin disease so so basically
there's a lots of reasons we can get
usually again low protein levels there
are over 200 proteins in the body seems
to me that's probably a whole lot more
than that actually some proteins change
dramatically with disease conditions and
others change only very little so there
are age-related changes in plasma
protein concentrations so basically you
have to take that and can into
consideration when you're dealing with
older animals most commonly in vet
medicine we perform three different
protein assays and that's total protein
that's the easy one that you can do
yourself any day in clinic albumin and
we do this of course in our automated
chemistry analyzers and then fibrinogen
and that's part of a if we're looking at
the clotting cascade that's one thing we
look at but it's a quick review of what
plasma proteins do
they are of course a large part of the
structural structural matrix of all
cells organs and tissues they maintain
osmotic pressure so they help to keep
blood in the blood vessels for instance
they serve as enzymes for biochemical
reactions and of course our bodies rely
on those enzymes in order to basically
work at all so very very important they
can act as buffers and acid-base balance
again very important because we need our
pH to be at just the right level or our
enzymes won't work and our bodies will
shut down plasma proteins are also
hormones some of them they function and
of course in coagulation of the blood as
I just mentioned with fibrinogen and
they defend the body against pathogenic
microorganisms you guys know all about
that from immunology and they serve as
carrier molecules for a lot of different
things in the plasma very very important
total protein so total plasma protein
includes fibrinogen total serum protein
does not and why is that well plasma
comes from unclouded blood serum comes
from carotid blood so if we allow the
blood to clot that will use up the
fibrinogen and there will be none in the
serum make sense right
there's a few things that can alter
total protein concentrations well more
than a few really but here are some
altered hepatic synthesis so the livers
not making as much protein as it should
often happens in the liver disease
altered protein distribution may be the
circulatory systems not working as well
and and the proteins not making it to
where it's supposed to be altered
protein breakdown or excretion again
usually liver maybe GI would be
responsible for that breakdown excretion
usually kidneys so also some GI so maybe
there's some issues with those organs
dehydration of course we have
dehydration that means the fluid portion
of the blood is less so then the
proteins are going to seem like they're
more so they'll be higher and then over
hydration the opposite happens and
proteins going to look low even if it
really isn't it's just there's too much
fluid in the blood
total protein measurement is really
valuable in determining hydration status
so I just talked about hyper protein
emia so elevated proteins being seen
with dehydration and then the opposite
with over hydration we can use this also
to screen patients with edema so that's
basically fluid buildup in the tissues
ascites and then you'll remember ascites
this free fluid in the abdomen diarrhea
weight loss hepatic and renal disease
and blood clotting problems there are
two primary ways that we test for total
protein the one that's really easy to do
in clinic is refractometer and that
estimates the refractive index of the
serum or the plasma you all know how to
use these you've done it in your lab
rotation and you've probably done it in
practice so it's fast it's inexpensive
it's accurate it's great so this is a
really great easy way to test total
protein the other ways a burette not as
common but it measures the number of
molecules containing more than three
peptide bonds in serum or plasma it's
simple it's accurate
again not usually used in clinic there
are other methods of testing that are
done in research and reference
laboratories you don't need to worry
about it there's you know
electrophoresis that sort of stuff
one kind of interesting thing is they
can do with electrophoresis if they can
find what type of protein they're
actually seeing so we've got albumin
here and then we've got globulin alpha
beta gamma globulin and gamma globulin
if you'll remember like immunoglobulin G
for instance very important with the
immune system albumin is one of the most
important proteins in plasma serum it
makes up about 35 to 50 percent of the
total plasma in most animals it's huge
so basically if we have hypo Prout
anemia it's usually because we're losing
albumin albumin is made in the liver
cells and hepatocyte s-- so again any
diffuse liver disease that's actually
injuring those cells or affecting those
Celes is going to result in a decreased
production of albumin renal disease you
can lose a lot of albumin through the
kidneys if you've got renal disease
dietary intake so sometimes like with
old cats with renal failure some people
put them on a low protein diet and that
will decrease their albumin as well
intestinal protein absorption so so if
the gastrointestinal system is not
necessarily working properly then
sometimes we get changes in levels of
albumin as well another really important
thing about albumin is that it is a
major binding and transport protein very
important in what we do for one thing it
maintains osmotic pressure of plasma
what that means essentially is that it
keeps the fluid component of blood in
the blood stream so it keeps our blood
pressure upright don't worry about the
photometric tab so there's some diseases
and conditions that are associated with
alteration in serum proteins can have a
quick look through these one thing I
wanted to mention actually before I
start talking about globulin this with
albumin is that because it is a
transport protein and I'm not sure if I
mentioned this down the line here on
this in this PowerPoint or not so I'll
just mention it now because it is a
transport protein for certain drugs then
if there isn't enough of it in the blood
stream then sometimes that alters the
transport distribution of drugs it can
also alter the amount of free drug
that's found in the in the plasma or the
serum and that can really cause trouble
in terms of the amount of active drug in
the blood stream but I think I do come
back to that later globulin so alpha
globulin are synthesized in the liver
primarily they are transport and they
transport and bind proteins there's life
of proteins high-density very low
density lipoproteins
these are alpha-globulin
beta-globulin they include compliment
so c3 c4 compliment is a very important
part of the immune system I know we
didn't talk too much about it but just
know that these are proteins that are
very important in the immune response
transfer and in ferritin so these are
responsible for iron transport he in
binding and fibrin formation and lysis
gamma globulin so I talked about these
just a couple of slides ago they're
synthesized by plasma cells they're
responsible for antibody production
right and immunoglobulins and animals so
I talked about IgG and there's of course
itd IgE iq8 IgM so all of these are
immunoglobulins these are gamma gamma
globulin so that's why when we do the
electrophoresis we can see what kind of
globulin s' we have in our bloodstream
and this is really important we know if
we could know potentially if we have low
immune function or high immune function
if we're fighting something very very
interesting there's also direct chemical
measurement of globulin sis is rarely
done so concentrations are estimated by
the difference between total protein and
albumin concentrations and that's
usually how we actually determine them
an alteration in the albumen globulin
ratio can be the first indication of a
protein abnormality so there's a range
that that should be in and and if you
run an automated blood test that's going
to show you that range so you can
compare the ratio to what it should be
and if we find we have actually more
albumin than globulin or vice-versa that
can really indicate where in the body
the problem might be so we actually
analyzed that ratio in conjunction with
the protein profile and a lot of the
pathologic conditions alter the the
ratio of albula albumin to globulin if
the concentrations are reduced in equal
amounts there'll be no alteration in the
ratio the most common cause of that
would be hemorrhage because you're
losing both types of protein equally
whereas through the kidneys you may be
losing albumin not globulin
you know basically one is typically lost
more than the other depending on where
the disease is except of course we think
in the case of hemorrhage
obviously this ratio is calculated by
dividing the albumin concentration by
the globulin concentration so in dogs
horses sheep and goats albumin is
greater than globulin this may be an
important BT any question it sounds like
the kind of stuff they'd ask you
so albumin to globulin ratio is greater
than one in dogs horses sheep and goats
and cattle pigs and cats it's less than
one so there is less albumin the same
amount or less than globulin in those
hams fibrinogen i've mentioned it's
synthesized by have pata sites again so
it's a precursor to fibrin and that's an
insoluble protein that forms the matrix
of the blood clots very important you
can form what blood clot without it so
if you have deep these five religions
you don't get or at least I don't get
much clotting fibrinogen makes up for
three to six percent but the total
process the plasma protein is not that
serum we've been over this why just a
note acute inflammation or tissue damage
might elevate fibrinogen levels the
values of fibrinogen are actually
calculated by subtracting the total
plasma protein value of a heated tube
from that of an unheated tube don't
worry about this too much some automated
analyzers can provide fibrinogen results
most of them don't but we can we have
ways of finding it if we need to a lot
of times it's sent to a reference
laboratory all right we're moving on to
liver so hepatobiliary assays a couple
of things about the liver first so the
liver is the largest internal organ in
the body it's not a complex structure
and very complex function and
interesting pathologic characteristics
actually so the liver is complicated
basically a large is complicated
it does a lot of things and when there's
liver disease there's all kinds of
trouble so what does the liver do well
it metabolizes amino acids carbohydrates
and lipids synthesizes proteins of
albumin primarily cholesterol plasma
proteins and clotting factors it digests
it absorbs nutrients it secretes
bilirubin or bile it eliminates and
detoxifies toxins and it catalyzes
certain drugs a lot of drugs actually so
very very very incredibly important and
again it does a lot of stuff so this is
where things get a little hairy
sometimes but I'll try to guide you
through in the simplest way I can all of
these functions are run by enzymatic
reactions and again that goes for most
of the body anyway as part of the powder
biliary system we have the gallbladder
so it's closely associated with the
liver as you'll remember from Anatomy it
kind of sits almost right in the middle
of the liver anatomically and
functionally it works of course together
with the liver its primary function is
storing the bile that's produced by the
liver if we have malfunctions in the
liver or gallbladder so in the biliary
tree we get jaundice low albumin we can
get problems with hemostasis and again
you think about that clotting factors
and and proteins and that sort of stuff
come from the liver hypoglycemia again
livers are very much involved in
controlling blood sugar levels hyper
lipoprotein emia have Hado
encephalopathy very very big deal if
there's something going on in your liver
or gallbladder so you may or may not
remember that the liver is divided up
into zones basically and the liver cells
are able to part mentalize damage so
that if one zone is damaged it won't
affect all liver functions so that's why
sometime
we'll see one component of the liver
that's not working or one function of
the liver that's just not not working
properly whereas the rest of it can look
fine and that can even happen with
significant liver disease there are over
a hundred tests available to detect
problems with the liver and again that's
because it is so complex and there's so
much that it does and no single test is
superior to any other it's just that
some of them are more appropriate to
certain conditions than others and
there's always tests that are being
developed to try to detect problems with
the liver sooner here are some of the
major patter biliary assays that we use
kind of every day so if we're looking
for enzymes that are being released from
damage directly damaged liver cells that
would be a LT probably our most common
one that we look for alanine
transaminase and then we have aspartate
transaminase as well and that's ast
those were the two that we're gonna look
at most commonly in practice in terms of
problems with cold stasis so that means
basically bile flow through the biliary
tree so if we're looking for enzymes
associated with that Alka fossa
so alkaline phosphatase very much the
go-to one for most clinics the other one
that we'll see a lot is GGT and that's
glama gluten will transfer a's so those
two GGT L Alka foss very commonly looked
at for cholestasis
and the long if that goes bilirubin and
bile acids so I just mentioned this in
the last slide bilirubin and bile acids
are commonly run in veterinary settings
of the liver function tests are less
sensitive and unfortunately these tests
often don't detect damage until
two-thirds to three-quarters of the
liver tissue is already damaged we can
look at albumin and cholesterol as well
in terms of production in the liver just
to get an idea of how well the livers so
a few notes on bilirubin
Billy Rubin is an insoluble molecule
it's derived for the breakdown of
hemoglobin by macrophages in the spleen
it binds to albumin for transport to the
liver just like a lot of things do the
liver cells actually metabolize that
bilirubin conjugated basically that
makes it able to be secreted in the bile
and the bacteria in the gastrointestinal
tract act on a bilirubin glucuronic and
they produce things called urobilinogen
x' and if you'll remember that's one of
those tests on your analysis that you do
urobilinogen czar then broken down and
excreted in the feces but they can also
be absorbed in the blood and excreted
through the kidney and that's why we're
looking at that value in your analysis
as well this is a nice complicated slide
it's actually extremely simple
simplified but I won't make you remember
it or anything if you want to you can
pause the video have a look at this it's
just it gives you an idea of how
bilirubin is metabolized within the body
measuring bilirubin can actually help us
pinpoint the cause of jaundice
we can determine the relative solubility
of bilirubin molecules and that can help
us to identify them where they came from
so it's pretty hepatic bilirubin so it
hasn't been conjugated by the liver yet
and it's bound to albumin so that
comprises approximately two-thirds of
the total bilirubin and the serum if
there are increases in that in that
unconjugated pre hepatic bilirubin then
that's usually indicating a problem with
uptake in the liver conjugated bilirubin
is measured directly and it indicates if
it's a height indicates bile duct
obstruction so the bilirubin is making
it into the liver it's getting
conjugated ready to be secreted or
eliminated from the body but for some
reason it's not making it through that
biliary tree so if we have increased
blood conjugated bilirubin well there's
a problem getting the bile out of the
liver bile acids are a really
great way to look at liver function so
this is not something that we're gonna
do in every animal but certainly if we
have an animal where we suspect liver
disease this is a really good test to
run bile acids serve many functions
among them they help us fat absorption
and they modulate cholesterol levels
they are synthesized in liver cells from
cholesterol they get conjugated don't
worry about that but then there are
secreted through the biliary system into
the duodenum where they can have their
effect
there's store to the gallbladder until
it contracts when an animal eats except
in horses horses are an exception to a
lot of things you'll see but usually
they hang out the gallbladder until an
animal eats then they're the golf
bladder contracts and then they make
their way all the way into the duodenum
once they go through the duodenum they
do their thing they reach the ileum then
they get transported back to the portal
circulation which brings them back to
the liver 90 to 95 percent of bile acids
are actually actively resorbed in the
ileum which is great so we recycle them
we can use them again right great the
remainder go out in the feces so there's
a little flow chart here that shows you
what all happens basically the bile
acids go from the liver to the gall
bladder into the intestinal tract most
of them go back to the portal
circulation and back to the liver some
of them end up in the systemic
circulation but most of them go back to
the liver and do their thing again some
of them go to the feces so they'll go to
the systemic circulation but this is
normal
postprandial so after eating is what
that means
serum bile acid concentrations are
higher than fasting concentrations that
makes sense because they only make it
into the bloodstream after the
gallbladder contracts and they help with
the fat absorption and then they go back
into the circulation or some of them do
so any process that interferes with the
circulation of bile acids results in
elevated bile acid numbers well that
makes sense
so they are also really great for
evaluating it's a major anatomic
components of the Pato biliary system so
what that means is that we can use this
test to look at anatomically and
physiologically how is the liver
actually working how do we do the tests
well we take two serum samples so we
fast the animal for 12 hours and then we
take a serum sample then we feed the
animal and then we take another serum
sample two hours later so we look at the
difference in the bile acid
concentration when an animal's fasted
and after eating horses we only take one
sample there are factors that can result
in false results basically inadequate
fasting we need that 12 hours in order
to get a good baseline food aromas can
actually cause some contraction of the
gallbladder prolonged fasting or
diarrhea can actually alter these
results as well so just some things to
keep in mind so say we have an animal we
do the bile acid tests they come back
elevated so this indicates the liver
disease but it doesn't tell us what
liver disease one of the most common
ones that we do this for is a congenital
portosystemic shunt so again this is
also sometimes called a liver shunt
usually what it means is there's a blood
vessel that's bypassing the liver so
instead of having all the systemic blood
go through the liver and get filtered
some of it bypasses the liver goes right
back into circulation and we get a toxin
buildup in the blood stream so that's
support of systemic shunt congenital
means the animal was born with it
there are acquired ones as well but
we're worried about the congenital at
this point chronic hepatitis so
hepatitis of course being inflammation
of the liver and so I mean there's lots
of things that could cause that a lot of
them viral and that kind of thing so
chronic hepatitis so we can have high
bile acids cirrhosis cirrhosis we
associate mostly with drinking so
alcohol and people
obviously most of them don't drink but
they can get cirrhosis for other reasons
we can get elevated bile acids with
cholestasis the biliary tree isn't
flowing normally for some reason and we
can definitely get these elevated cancer
of course and then the other thing about
bile acids again they're unspecific for
the type of liver problem it's a
screening test basically we're asking is
the liver functioning normally and if
not then we'll go on and try to figure
out why we already looked at this just
in case you wanted a review here's your
beautiful flowchart you can pause and
look and enjoy
let's talk cholesterol for a few minutes
so it's a plasma lipoprotein that's
produced primarily in the liver but it's
also ingested in food and we know that
in people they always say oh don't eat
too many eggs because your look at high
cholesterol well actually that's not
true but cholesterol is something that
we worry about a lot of people we don't
worry about it so much in animals except
in very a very select few disease
processes cholestasis causes increased
cholesterol in some species again
cholestasis
the bile is not flowing properly through
the biliary tree so that can increase
the cholesterol for some species it's
not actually very well characterized in
veterinary species but here's the thing
we sometimes use it to screen for
hypothyroidism hypothyroid dogs will
almost always have elevated cholesterol
so if we have a dog that looks like it
could be hypothyroid it's kind of
overweight it's kind of lethargic it's
got bad skin whatever and it has high
cholesterol maybe we should run a t4 TSH
on that dog just to see do we actually
have potentially hypothyroidism so
that's one of the most common uses for
cholesterol testing in vet medicine
cholesterol also does not cause grossly
like quemic plasma after eating so if
you're spinning down a sample and an
animal who's eaten in the last you know
hour to
and you notice that it's like quemic
it's not cholesterol let's quickly talk
about enzymes that are was released from
damage to parasites I mentioned these
before but again as liver cells are
damaged certain enzymes do leak into the
blood and we can measure these when they
start to leak into the blood right so we
get our alt alanine transaminase our ast
aspartate transaminase and dehydrogenase
enzymes I D and G LDH again these are
probably not ones that you're gonna see
commonly used in practice these are
reference laboratory tests that can be
sent away for in certain situations and
your vet will decide that again the only
readily available tests are alt and ast
I would say alt is probably our most
commonly looked at one the major source
of alt is the hepatocyte makes sense
it's found free within the cytoplasm of
hepatocytes so that when there is liver
damage within 12 hours you can see it
leaking into the bloodstream so we'll
see elevated serum or plasma levels
within 12 hours its alt is considered a
liver specific test and dogs and cats
and primates but horses ruminants pigs
and birds don't have enough alt to be
considered a liver specific test so if
you're dealing with those species not as
useful to you but again for dogs and
cats which most of you will deal with
alt is a really great go to test for
liver it can be elevated by damage to
renal cells as well cardiac muscles
glial muscle of the pancreas so bear
that in mind so the results does need to
be taken with a bit of a grain of salt
if there's known problems in those areas
again the major source is the liver
and this is considered a liver specific
test in dogs and cats and non-human
primates if you happen to be working
with us it's a screening test so we it's
not specific myth enough to identify the
liver disease see sounds familiar right
it's like every liver test
but alt very commonly used as a
screening test for liver ast also
president hepatocyte it's in the
cytoplasm and it can be found bound to
the Medical conjul membrane and it takes
more severe liver damage to release AST
the membrane bound stuff at least so
levels will rise slower than alt but
they were returned to normal faster
interesting is he is unfortunately also
found in other tissues red blood cells
cardiac muscle skeletal muscles kidney
and pancreas so common causes for
elevation could be not just in the liver
it could be elsewhere but liver disease
is probably the most common one but
muscle inflammation or necrosis could be
a big one and hemolysis so keeping that
in mind aspartate transaminase AST just
has to be has to be considered in
conjunction with the clinical picture
which is of course true of all these
tests cold stasis so I've mentioned this
several times but here we go again bile
duct obstruction that's ultimately what
it comes down to right the bottle can't
make it through the biliary tree the big
one that we look at in that medicine is
alkaline phosphatase AP or alpha this is
present in osteoblasts as well so
osteoblasts you'll remember our cells
that produce bone chondroblasts cells
that produce cartilage intestines and
placenta and liver cells so several
places this can come from this is not a
great specific test but in conjunction
with other tests that can point towards
coal stasis GGG is found in many
different tissues and comes primarily
from the liver as well cattle horses
sheep goats birds they have higher GGT
normally than dogs and cats so again
that is to be borne in mind working with
those species and elevated liver levels
of two
do have it with liver disease especially
if it's an obstructive liver disease
again obstructive liver disease we're
talking about cholestasis
so just a summary of what we've gone
through so far total plasma proteins
include fibrinogen total serum proteins
do not albumin makes up 35 to 40 percent
of total protein liver function tests
measure substances produced by the liver
unconjugated bilirubin comprises
approximately two-thirds of the total
bilirubin and most species bile acids
aid in fat absorption and modulate
cholesterol levels elevated serum bile
acids do indicate liver diseases not
specifically though and if we're looking
for leakage enzymes alt and ast are the
ones we'll will evaluate first alt and
ast are the most common performed liver
tests