You might have heard of the term direct bilirubin and indirect bilirubin. What
does that mean? I think most of you must be knowing that direct bilirubin means
conjugated bilirubin and indirect bilirubin means unconjugated bilirubin.
But why they are named so? Today I will explain you the exact reason behind
this naming. Right, hello I am Dr. Aizaz from Medicovisual.com welcome to this
visual lecture. So let's suppose this is a macrophage you know in spleen and even
outside the spleen when RBC's are broken hemoglobin is released. Hemoglobin
consists of heme and globin. Globin is basically polypeptide chain. Globin chain
is digested and converted into free amino acid and here the heme which comes
into macrophages, which is taken up by macrophages, it is converted into
bilirubin through a multi-step pathway. Right, why so? Because heme is a toxic
substance it can cause direct cell injury it can even lead to multi organ
damage especially it can cause kidney damage and can lead to acute kidney injury.
Right, so macrophages thankfully convert, they convert heme into bilirubin.
Right, now this bilirubin should be excreted out of the body through feces.
It is sent to intestines through blood and then it is lost in to feces. Right,
now when it comes, bilirubin comes into blood here there is a problem actually
bilirubin is a non-polar substance it's a non-polar, hydrophobic, lipophilic
substance. You know blood is 90% water. It's an aqueous medium so when in
aqueous media, when in this water oily substances, bilirubin is added it
coalesce together, it aggregates together and it starts sticking to the walls of
of blood, blood vessel I mean. Right, so it is just like as if droplets of water,
droplets of oil they start floating on water because that's a lipophilic
substance. So here there is a problem and the solution for this problem is that we
have special transport vehicles for this. Right, what is the name of this
transporter? This albumin, albumin forcefully takes up this bilirubin and
it can then transport to the intestine. Right, if albumin was not there it will just
keep sticking, bilirubin will just keep sticking scared of water, it will
just keep sticking to the walls of blood vessels. Thankfully albumin is there to
take up this bilirubin in hydrophobic environment and to transport it to
intestine. Now when it will transport it to intestine again the problem is the same.
Intestine is full of water. Right, so when it will be sent into the intestine, when it
will be unloaded into intestine again here it will be aggregated and it will
coalesce together and again it will stick to the wall of the intestine and it won't
be excreted. So what should be done? One thing that can be done is there this
albumin it takes up this bilirubin and it then drops it into feces and in this
way albumin will also be lost in to feces. Of course we don't
our precious trucks, our precious transporter to be easily lost into feces.
So again this is a problem when we have our friend liver.
So liver solves the problem. Liver says don't worry just give this bilirubin
molecule to me I will do something with this molecule and then this molecule
will be lost into intestine, into feces without need of losing this albumin. so
what liver does this reminds me of something when I was in high school
there was a very wise teacher what she did is what she used to do is that, there
were, of course, some academically weak students in our class, she paired up
those academically weak students with some brilliant hard-working students.
Right, then when these weak students were paired up with those brilliant students
they also started working hard and their grades went up so that's the idea which
this liver follows. The liver is like a wise teacher. I think what liver does is that
liver pairs up, liver conjugates this bilirubin with this weakly polar, this
weakly polar bilirubin with a strongly polar substance and what is this
strongly polar substance? That is glucuronic acid. So liver attaches two
molecules of glucuronic acid with bilirubin and now this bilirubin becomes
bilirubin diglucoronide. Now question that here this was bilirubin
which, which was loaded on these trucks is this bilirubin conjugated bilirubin
or this bilirubin is conjugated? This glucuronide bilirubin, this
bilirubin diglucuronide is conjugated bilirubin. Actually when I was
kid I thought that as soon as bilirubin is attached with albumin it is
conjugated bilirubin. It is not conjugated bilirubin it is unconjugated
bilirubin because it is not chemically bound with it, it is not covalently bound
with it. This bilirubin with which it is covalently
conjugated with the two molecules of glucuronide this bilirubin is called
conjugated bilirubin and this bilirubin is unconjugated bilirubin which
was loaded on albumin. Right, so now that this highly hydrophobic substance is
bound with glucuronic acid two molecules of glucuronic acid now it has become
slightly polar, and now it has become slightly polar or hydrophilic now it can
be easily lost into intestine without any help from albumin and now it will be
lost into, it will be secreted into the intestine through bile, bile duct. Right
and it will be lost in two feces. Now let me tell you an experiment. Now let's
suppose this is a container and in this we add plain water and in this plain
water when we add this conjugated, this conjugated bilirubin it will be easily
dissolved, why? Because it's a polar substance. Right, now let's take a
container and in this container now we add a special substance into this
container and the substance is called Diazo-sulfanilic acid. For medical
students I don't think you need to remember the name, the
chemical name but at least remember that this is called this substance is called
Van Den Bergh's reagent on the name of a scientist Abraham Vandenberg who was
pioneer of this technique which I am explaining right now. Right, so this reagent
is added, this reagent is added into water and now in this solution if we add
conjugated bilirubin, this conjugated bilirubin and this reagent it will
quickly, swiftly react with each other and it will form a purplish substance a
purple substance that is, that is chemically called
azo derivative of bilirubin, right, and due to this presence of, due to
formation of this purplish, this purple colored pigment, this purple colored
substance, this whole the solution will turn purple in color. Right, the color will
turn purple. Now the intensity of color it depends upon, it is directly
proportional to the concentration of bilirubin. If more bilirubin is there
the color will be more intense if very less bilirubin is there, I mean conjugated
bilirubin color will be less intense. Right, now depending upon on the basis of
the intensity of color, on the basis of the intensity of purple color we can compare
it with a standard and we can estimate the concentration of bilirubin present.
Of course, if color is more intense it means more bilirubin is present. Right, so
we can estimate the bilirubin by this method. Now because this bilirubin
directly reacted with this reagent, it directly reacted with Van Den Bergh's
this reaction is called direct Van Den Bergh's reaction, reaction direct
Van Den Bergh's reaction and this conjugated bilirubin is called direct bilirubin.
Right, now let's repeat the experiment again. Now in water first we add, what is
this reagent Van Den Bergh's reagent and now rather than adding, adding conjugated
bilirubin now we add unconjugated bilirubin, free bilirubin. Now as soon as
this free bilirubin is added scared of water it will quickly come together, the
molecule will come together and hug each other. Right, so it will, they will
coalesce together and they will form this oily droplet on the surface of
water so now this reagent can it react with this? It can react but it will react
very slowly. Right first it will react with this outer layer of molecule then
it will go inside, actually it has become like this, there, there's this is an outer
layer then inner layer of molecule then another inner layer of molecule and
ultimately the core. So first molecules will this molecule of
Van Den Bergh's reagent first they will come and react with this outer layer then
this inner layer and then this inner layer and so on reaction will be very
very slow. Here in case of direct bilirubin the molecules were spread evenly,
molecules of conjugated bilirubin were spread evenly surface area was quite
high, surface area was large because of this it quickly, swiftly reacted without
adding any catalyst without doing anything but here reaction will be very,
very slow. Right, so in this when we add a catalyst, and catalyst for this reaction
is methanol, when we add few drops of alcohol,
methanol, 50% alcohol, it acts as a catalyst and now it will quickly react and color
will turn purple. Now you see in case of unconjugated bilirubin the reaction was
very, very slow unless we added a catalyst. methanol so reaction proceeded
indirectly after addition of the catalyst, after addition of methanol so that's why
this type of reaction is called indirect Van Den Bergh's reaction and the unconjugated
bilirubin which undergoes this reaction is called indirect bilirubin. Right, now
let's test your knowledge. Let's suppose this is a container and in this
containers plain water is added and now first we had let us say this reagent
Van Den Bergh's reagent and then I add some bilirubin molecules I don't know which
bilirubin is this and now when bilirubin, this bilirubin is added within
few seconds the color turns purple, the color turns purple which bilirubin was
this? Direct bilirubin. Now when let's suppose concentration
according to intensity when we estimated the concentration of bilirubin it turned
to be let's suppose 7. Right, now we did something amazing.
Now, we in this solution, we added methanol and when we added methanol
the intensity increased. Right, color, the intensity increased and now when we
checked, when we estimated the bilirubin concentration it turned out to be
about ten. So this concentration ten of bilirubin is which bilirubin conjugated
or unconjugated? Unconjugated. No it is not unconjugated. What is it? Can you tell?
It is total bilirubin. Actually what happened? This was actually a mixture of
bilirubin, it was mixture of direct and indirect. Let's forget about direct, for
sake of simplicity it was a mixture of conjugated and unconjugated bilirubin.
Initially what happened? Initially immediately when bilirubin, this mixture
was added immediately conjugated bilirubin reacted and value turned out
to be seven. Right and unconjugated bilirubin remained behind it didn't
react. Right, but when methanol added, methanol was
added, even unconjugated bilirubin also reacted and intensity increased because
unconjugated bilirubin also reacted intensity increased and now this total
intensity ten is actually the concentration of total bilirubin. From
this total bilirubin we can estimate, we can calculate the indirect bilirubin, how?
Total bilirubin - conjugated bilirubin. I think it's very easy to remember that
ten is total bilirubin right and out of this the initially that reacted was
seven so indirect bilirubin is about three. Right, indirect bilirubin is so
indirect that we do not directly measure it. We indirectly calculated from total
bilirubin and you clear about the equation that how, from where this
equation came. Are you really clear? So in a nutshell the
direct bilirubin is conjugated bilirubin and indirect bilirubin is unconjugated
bilirubin but now you know why. I have seen many students and even health care
professionals remembering it like this, cramming it up like direct bilirubin
conjugated bilirubin and indirect bilirubin the unconjugated bilirubin and on the
day of exam they again forget it. You don't need to memorize this my friends
it's very logical you just need to understand the concept and then studies
become very easy for you. Right, so this was about the Van Den bergh's reaction and
direct and indirect bilirubin. Thank you so much for watching this video. If you
like this video please give a thumbs up and please do subscribe to my
channel and do check out my Facebook group also please give your precious
comments regarding this video. Thank you so much.