Metabolic lab summary

Dr.Abdullah Hamamdeh

By: Dareen Njoom & Duha maslamani

Lecture 2

Spectrophotometer:

1.Definition

2.Components

3.Transmittance

3.Absorbance

4.Expirement

Definition of spectrophotometer:

Is an Instrument applied a light with specific wave length into a sample tube, and according to the
type of light that is transmitted or absorbed within the sample tube or through the sample tube we
can detect light type.

Transmittance: the amount of light that is transmitted through the sample tube.

Absorbance: the amount of light that is absorbed within the sample tube.

Components of the spectrophotometer:

1.light source (source of Photons): give beams of light.

2.Monocrometer (Prizm): responsible to divide the light into specific wave length.

3.Cuvette: contains the solution (solute+solvent)

4.Detector: give digital number (for the absorbed light)

 I0: the intensity of the applied light, I: the intensity of the transmitted light.

Incident light can be

reflected off, absorbed by, or transmitted through a sample.

What are the factors that alter the amount of the light that is absorbed within the sample tube?

Answer:

1. concentration of the solute within the sample tube.

What is the relationship between the amount of the absorbed light and the concentration of the
solute? (‫)عالقة طردية‬

2. cell pathlength (cuvette ( (1cm)

What is the relationship between the amount of the absorbed light and the cell pathlength?
(‫ )طردية‬the contact time between the photons and cuvette is increased due to increase the
length of the cuvette.

What is the logarithmic relationship between the Absorbance &the Transmittance?

T=I/I0 X100

A=-LogT

Beer's lambert law:

A=e XL (cell pathlength)X[solute]

Linear relationship (‫)طردية‬

e : extinction coefficient is known (1/Mcm)

L:1cm

What is the relationship between lambda (wave length) and the amount of the absorbed
light?
There is no relationship between them. the maximum light of absorption occur in specific
wave length, but does not effect the amount of the absorbed light.

Reference blank: cuvette contains everything found in our sample solution except the substance we are
trying to analyze or measure (solute).
If we applied a light into a sample tube every thing in this sample tube (cuvette)will absorb the
light, for instance our solute (violet (dye)) & our solvent (distil water), the dye will absorb the light in
specific amount, and the distil water will absorb the light in specific amount. The transmitted light reach
to the detector and the detector will give us a digital number for the amount of the absorbed light inside
the cuvette.
The point of the experiment is to measure the amount of light that is absorbed only by the solute (dyes),not
to measure the amount of light that is absorbed by the solvent.
The reference blank is used to help in ignoring any light absorbed by the solvent and measures only the
light absorbed by the solute.

The experiment:
The solute (dye) the solvent is distil water
In our experiment we will:
use different dyes
Apply specific wave length on these dyes
Detect the maximum absorbance of the light occur in which wave length.
Optical density: is another name for maximum absorbed light

Dareen Njoom
Liver function test (LFT):
Lecture3
Enzymes:
-Definition:
catalyst that speed up the chemical reaction.

-where do they work in the chemical reaction?

A-B +C → A- - B-C →A +B-C
Reactant: A-B +C
Transmitted state: A- - B-C
Product: A +B-C

Delta G= Energy of Transmitted state-energy of the

Reactant.

Enzymes reduce the activation energy.

In the presence or absence of enzymes the chemical

reaction will occur, the only thing will be change or alter
is the velocity of the chemical reaction.

Enzymes we will work with:

-Phosphatase: cleaving of phosphate group
- Transaminases

In general the liver function tests will be divided into three parts:
1.Tests reflect the function of the cellular unit of the liver (hepatocyte)
-ALT
-AST
(Transaminases)
2.Tests reflect the function of the biliary ductal system
-ALP (Alkaline phosphatase)
-ACP (Acid phosphatase)
3.Tests reflect the function of the synthetic function of the liver
-Bilirubin (at some degrees may reflect the biliary ductal system)
-Albumin

Test reflect the function of the biliary ductal system:

-ALP (Alkaline phosphatase):

1.catalyste
2.Removes phosphate group
3.works better in alkaline media
The normal range for ALP is approximately (44-144 unit/liter)
This enzyme released from different parts of the body into the serum, mainly
from:
1. epithelial lining of the biliary ductal system (the most important)
2.small intestine (we can note postprandial elevation (means after eating)), so
when we are going to do approach for a patient with normal ALP level, the
patient should be in fasting state.
3.placenta (physiologic elevation during pregnancy)
4.the bone (osteoblasts), so another physiological elevation of alkaline
phosphatase is during childhood.
-Physiological elevation:
Pregnancy & childhood
-pathological elevation:
Divided based on the amount of ALP in the serum.
Bigger than 5Xnormal range
less than 5Xnormal range

upper than 5Xnormal range

What causes upper than 5Xnormal range?

Any problem with the previous sources of ALP.
 1Osteomalacia: softening of the bone in elderly people, abnormality of
mineralization of the bone.

Deficiency of vitamin D → accumulation of osteoid (is the unmineralized

collagen)→softening→increase the risk of fracture.
Osteomalacia is not osteoporosis, the total bone mass does not change in
osteomalacia , but change in osteoporosis , the bone more thin & the bon is
mineralized.
2.Rickets: occur in childhood, bending in the bones occur and the pathophysiology for
this disease is the same of osteomalacia.
3.Paget's disease of bone (osteitis deformans): over growth of the bone at focal
site, mainly (skull, spine), it is believed that, there is abnormality in osteoclastic
activity (osteolytic).
The patients with Paget's disease enter three phases of bone remodeling.
What is THE most common cause with asymptomatic elevation of ALP in elderly?
The answer is Paget's disease.
4.Cholestasis: impairment of out flow of the bile.
Normally: bile produced in the liver→biliary ductal system→some of this bile stored
in the gallbladder and some of it reach duodenum.
impairment of out flow of the bile due to:
Internal abnormality: stones→ abnormality in the pathway→stasis of bile within
biliary ductal system→ elevation in ALP
External compression: obstruction by tumor→ prevents or block the outflow of
the bile→ stasis→ elevation in ALP

2. less than 5Xnormal range

1. renal bone disease (renal osteodystrophy)
2.hepatitis
3.inflammatory bile disease.
4.healing fracture.
ALP consider as a marker for bone forming.

Causes of decreased plasma ALP:

 1. Cretinism: Congenital hypothyroidism, is a severe deficiency of thyroid hormone in
newborns
2. Vitamin c deficiency
3. Decrease in the phosphate level in the body.

-ACP (Acid phosphatase):

1.catalyste
2.Remove phosphate group
3.works better in acidic media
Total ACP is exist in the circulation, the sources for Total ACP :
-prostatic in males: from prostate gland
-non-prostatic: bones, liver, spleen& kidney
To measure prostatic ACP:
Total ACP- non-prostatic
There is another test is used to detect prostatic cancer instead of ACP test, is
prostatic specific antigen.
What are the causes lead to elevation of ACP level in the body?
1. Prostate CA
2. Multiply myeloma
3. Sickle cell disease
4. Lysosomal storage diseases
ACP works on
Alpha-naphtholphosphate(substrate, colorless) +H2O by ACP→ alpha-
naphthol +Pi
Calculate the absorbance
immediately
After 1 min
2 min
3min
As The time moves on [product]→ [color]→ absorbance

The experiment:
The substrate for the reaction is nitrophenylphosphate (colorless)
The source of ALP is serum

Nitrophenylphosphate +H2O by (ALP)→ nitrophenol (yellow)+Pi (hydrolytic reaction)

-Calculate the absorbance
Immediately After 1 min
2 min
3min
As The time moves on [product]→ [yellowish color]→ absorbance

Dareen Njoom
Lecture4&5

Note,most of these details of liver 's components just to understand how the liver
do its function, not for the exam.

-Where is the liver located in the human body?

In the right upper quadrant of the abdomen.
The liver makes up 2.5% of the total body weight.(1.2→1.8 Kg)
-What are the functions of the liver?
1.glycogen synthesis. Extra glucose after a meal will be converted into glycogen and
stored in the liver. (prevent hyperglycemia)
2.gluconeogensis or glycogenolysis. trauma or stress may lead to drop of glucose level
in the blood, so The liver compensates for the deficiency with sugar by this pathways.
3.detoxification of the toxins and the bacterial enzymes.
If there is any toxins in GI(splanchnic circulation )→ the drainage of those organs go to
the liver→ the liver(here detoxification occur in order to prevent toxins from reach the
vena cava)→ general circulation (vena cava system).
splanchnic circulation: spleen, pancreas & stomach, the Venous drainage for those
organs should reach to the liver.

4.The functional unit of the liver is hepatocyte, which composed of:

-RER: responsible for plasma proteins production (eg, albumin which is very important
for maintaining the oncotic pressure and intravascular volume)
If there is liver abnormality→decrease in the synthetic function of the liver→decrease
the production of the albumin→that leads to extravasation of the fluid into the
interstitial system→edema
-SER: responsible for lipids and enzymes production, these enzymes catalyze estrogen.
This is very important in patient with liver failure.
What is the clinical picture for this patient?
 Eg, liver cirrhosis. Decreases in liver function→ decreases the production of the
enzymes that work on the estrogen→increase estrogen level in the blood which leads
for two things:
1.spider angioma: dilatation of the small arterioles under estrogen effect.
2.gynicomastia: increase in the size of the breast tissue in males.
5.the liver produces coagulation factors.
6.produces bilirubin.
7.produces lipoproteins
What are the blood supply to the liver?
There is dual blood supply for the liver
-Portal vein: is the drainage of the splanchnic circulation (union of splenic vein &
superior mesenteric vein) (70-80% mostly deoxygenated blood to the liver)
-Hepatic artery: the origin for this artery is from celiac trunk (20-30% mainly oxygenated
blood).
The liver is divided into multiple lobules, and these lobules have hexagonal shape.
The hepatic artery and the portal vein give branch for each corner for the hexagonal
shape.
Hepatic sinusoid: it is the union between the branches of the hepatic artery and the
portal vein from the periphery to the center (central vein)
Where are the hepatocyte located inside these lobules?
In between the two hepatic sinusoid.
Between the hepatocyte and the hepatic sinusoid there is a space called space of Disse.
Un conjugated bilirubin exit from the sinusoid to space of Disse and binds to its receptor
on the hepatocyte where the conjugation process occur and this conjugated bilirubin
goes to the biliary ductal system then it is released in the gastrointestinal system.
So the bilirubin direction is opposite to that of the hepatic sinusoid (from the center to
the periphery).
The portal triad on each corner of the hexagonal lobule composed of:
1.branch of the hepatic artery
2.branch of the portal vein
3. conjugated bilirubin

Tests reflect the function of the cellular unit of the liver (hepatocyte)
Transaminases: (amino transferase)

catalyst
 transfer the amine group from one molecule to another

AST: aspartate transaminase

ALT: alanine transaminase

What are the characteristics for the chemical reactions that we will add to them the transaminases?

Contain:

1.donor (have amine group) Amino acid

2.recepient for the amine group (keto acid : alpha ketoglutarate)

3.bidirectional (reversible)

AST or (GOT: glutamate oxaloacetate transaminase)

cellular enzyme presence in two isoforms of AST:
1.cytoplasmic: the elevation in this isoform indicates moderate cell injury.
2.mitochondrial: the elevation in this isoform indicates sever cellular injury.
Organs release AST
1.liver
2.kidney
3.skeletal muscle
4.heart

Donor: Aspartate
Recipient: alpha ketoglutarate
Bidirectional
AST transfers the NH2 from the A.A to the keto acid
From Aspartate to alpha ketoglutarate

Aspartate converted into keto acid (oxaloacetate)

Alpha ketoglutarate converted into amino acid (glutamate)
oxaloacetate will spontaneously enter another reaction in presence of NADH and gives malate &
NAD+ by malate dehydrogenase enzyme.
 ALT or (GPT: glutamate pyruvate transaminase)
Mainly produced by which organ?
Liver
ALT is more specific and more sensitive for liver diseases

Donor: Alanine
Recipient: alpha ketoglutarate
Reversible
ALT transfers the NH2 from the A.A to the keto acid
From alanine to alpha ketoglutarate
Alanine converted into Keto acid (pyruvate)
Alpha ketoglutarate converted into amino acid (glutamate)

+ alpha ketoglutarate by ALT → pyruvate + glutamate

Pyruvate will spontaneously enter reaction in presence of NADH and gives lactate & NAD+ by lactate
dehydrogenase enzyme.
From which cycle we can get lactate?
From Cori cycle

In the transaminases reaction reactions there are two constant in the reactant and in the product:

Alpha ketoglutarate in the reactant

Glutamate in the product

How can I distinguish if the patient suffers from problems related to the liver or another organs in the
body?

That depends on the ALT/AST ratio

Normal ALT/AST ratio =1

If ALT/AST <1, this means there is AST more than ALT in the blood, and as we mentioned before the AST
released by different organs in the body (liver,kidney,skeletal muscle, heart).so, this patient may has
myocardial infraction, because the heart is one of the organs which produce AST, high AST level may
indicates muscular dystrophy, or metastatic carcinoma.

If ALT/AST> 1, this indicates a problem in the liver, because this enzyme produced mainly from the liver.
Eg, Hepatitis, Cholestasis (accumulation of the bile) ,IMN (infectious mononucleosis, caused by Epstein-
Barr virus (EBV))

If AST/ALT=2/1, this indicates alcoholic liver disease.

Now we are going to talk about bilirubin:

Total bilirubin= 0.2-1.2 mg/dl

Total bilirubin=unconjugated bilirubin (direct) + conjugated bilirubin (indirect)

If total bilirubin>1.2 mg/dl, hyperbilirubinemia.

When will the symptoms appear for hyperbilirubinemia?

When total bilirubin>or=2.5 mg/dl, patient will produce symptoms and signs.(jaundice (yellowish
discoloration for four sites in the body: 1.skin 2.sclera 3.mucuse membrane (eg, oral cavity) 4.interstitial
fluid)

Not all hyperbilirubinemia=jaundice the total bilirubin should>or=2.5 mg/dl.

The half life of red blood cells = 120 days. After 120 days red blood cells will divided into its main
components, because red blood cell lack of nucleus so, they lack the ability to produce and regenerate
their own cytoskeleton.

What are the main components of red blood cells?

1.proteins Both of them proteins and

2.membranes membranes will enter the recycle of
the amino acid

3.Hemoglobin: composed of two main component:

1.globin: enter the recycling process of the amino acid

2.heme: by hem oxygenase will divided into two parts:

1.ferric (Fe3+)
2.protoporphyrin (4 pyrrole rings)

The pyrrole rings made circular architecture, if this structure change and being linear the product called Biliverdin,
biliverdin by biliverdin reductase enzyme will converted into unconjugated bilirubin (UCB) and the bilirubin will
go to the general circulation from RBCs.

The ultimate source or the main source of Unconjugated bilirubin is destroyed RBC (The only source for
unconjugated Bilirubin)

The features of unconjugated bilirubin:

1.highly toxic

2.Apolar

3.very large in size

4.water insoluble

According to the bad features of unconjugated bilirubin, should it be passed through the circulation freely?

It should not pass through the circulation freely, because it could deposited in multiple tissues and causes
destruction. And this UCB can cross blood brain barrier BBB and causes neurological abnormality.
So, the unconjugated bilirubin bind to proteins. And the liver is the main organ which produce proteins.

The main site in the liver which produces plasma protein is hepatocyte RER, the hepatocyte produces BBP
(bilirubin biding protein)
RBCs produce UCB→ go to the circulation
Hepatocytes produce BBP→
UCB-BBP→hepatic artery or the
portal vein→ hepatic sinusoid→exit
the capillaries→space of
Disse→receptors for the BBP on the
hepatocytes→BBP give the
hepatocyte the UCB and go back to
the circulation.

Inside the hepatocyte: UDP-

glucuronide transferase ( ‫الدكتور حىك‬
‫)كتي مهم‬
‫ر‬
The role for this enzyme is to add
glucuronic acid to UCB→converted
into conjugated bilirubin.

What are the features for conjugated bilirubin?

1.water soluble (could be excreted by the kidney)

2.polar

3.small in size

4.less toxic than UCB

There are specific transporters, which transport the conjugated bilirubin to the biliary ductal system.

(Center to periphery)

Biliary ductal system:

There is intrahepatic biliary ductal system& extrahepatic biliary ductal

system.

Right hepatic duct (RHD) + left hepatic duct (LHD) =common hepatic duct
(CHD)

common hepatic duct (CHD) + cystic duct (from the gallbladder) = common
bile duct

common bile duct + pancreatic duct→ open in the second portion of

duodenum (the conjugated bilirubin released here)
inside the intestine the conjugated bilirubin converted into urobilinogen.

Urobilinogen by intestinal normal flora converted to stercobilinogen (responsible for the color of the
stool)

Some patients have white stool→ this indicates there is something obstruct the common duct→
prevents conjugated bilirubin from reach the duodenum and thereby prevents conversion of
conjugated bilirubin into urobilinogen and stercobilinogen then there is no color for the stool.

What are the causes of increase the bilirubin in the blood?

Hyper Unconjugated bilirubin:

1.excessive hemolysis of RBCs

2. there is small amount of BBP or some drugs bind to BBP and prevent UCB from binding to BBP(eg,
sulfa drugs)→ increase the amount of UCB in the circulation.

3. deficiency of UDP glucuronide transferase. There are three diseases according to the degree of the
deficiency:

1.milde: Gilbert syndrome

2.moderate: crigler najjar type 2

3.sever: crigler najjar type 1

Hyper Conjugated bilirubinemia:

1.any problem with the transporters which transport the conjugated bilirubin to the biliary ductal
system, this divided into two diseases:

1.Dubin Johnson

2.Rotor syndrome
2.obtruction of the biliary ductal system (cholestasis)

- extrahepatic biliary ductal system obstruction

-intrahepatic biliary ductal system obstruction:

extra tumor will cause narrowing for the biliary ductal system.

Stones

Strictures. Eg for diseases related to strictures: Primary Biliary Cirrhosis PBC

Primary sclerosing Cholangitis PSC

These two diseases are autoimmune diseases.

The high level of bilirubin can be divided in this way:

Prehepatic causes: (causes high level of UCB)

1. hemolysis of RBCs
2. some drugs bind to BBP and prevent UCB from binding to BBP

Hepatic causes: (may cause high level of both UCB & CB )

1. deficiency of UDP glucuronide transferase

2. any problem with the transporters of conjugated bilirubin
3. intrahepatic biliary ductal system obstruction

Post hepatic causes (causes high level of CB)

1. extrahepatic biliary ductal system obstruction

TB=0.2-1.2

If TB>1.2, what are the symptoms and signs for these patients?

And, how to distinguish if the patient has hyper CB or hyper UCB?

Answer:

If the TB>1.2, should do CB test, if CB >20% of TB. I will think about the causes make CB level
high.

If CB<20% of TB. I will think about the causes of make UCB level high.

For example, if the TB=2 (2>1.2, CB test is required in this case) CB=1 (1=50%of TB), CB >20% of
TB. I will think about the causes make CB level high.

Dareen Njoom
 Lecture6:
*Total plasma protein:
FIRST: distribution of fluid within our compartment:
Total body water= 60% of total body weight
E.g.: A person weighing 72 kg, How much is the total body water? 42L
The 42 L will be distributed within our compartment as:
2/3 Intracellular fluid (28L)
1/3 extracellular fluid (14L):1. Interstitial fluid (3.5L)
2. Intravascular fluid (10.5L)
*What are the forces that control the movement of fluids in our component:
Hemodynamic forces or Starling forces:

1. hydrostatic pressure.
2. Oncotic pressure ( the other name is :colloid osmotic pressure)
* at arterial side:1. hydrostatic pressure=35mm
2. Oncotic pressure= 25mm
3.The net filtration pressure (out)=10mm (hydrostatic pressure ‫(لصالح ال‬, it pusher fluids from
intravascular to interstitial), this pressure is determined by the cardiac output ‫كل مكنا اقرب عىل‬
)‫ال بضخها القلب‬
‫فه بتتحدد بكمية الدم ي‬،
‫اكب ي‬‫)القلب كل مكان ر‬

*At venous side: 1.hydrostatic pressure=15mm (Less because it is away from the heart)
2. Oncotic pressure= 25mm (constant)
3. The net filtration pressure (in)=10mm (puller to fluids intravascular)
* excessive fluid within the interstitial: 80% return to Intravascular at the venous side, 15-20%
will return to the circulation from the lymphatic system.
if there is an excessive accumulation of the fluid within the interstitial, what we call this?
Pressure dependent Edema (at lower limbs)
what are the causes of the edema?
1.increase in hydrostatic pressure at the arterial side.
2. OR decrease the Oncotic pressure at the arterial side.
(Exactly the opposite on the venous side)
3. lymphatic obstruction.
*how I can distinguish between the edema caused from lymphatic obstruction or from heart
failure?? Heart failure cause pitting edema.

*Oncotic pressure: the other name is:colloid osmotic pressure, why? Because it depends
on the concentration of the protein in our intravascular component.

SO the total plasma protein=6-8 g/dl

What are the types of protein in the circulation:
1.Albumin= 3-5 g/dl
2.globulin= 1.5-2.5 g/dl
3.fibrinogen= 0.25-0.5 g/dl (in coagulation cascade with Prothrombin from liver)
The most important function of these proteins is to maintain the fluid within the
intravascular component (so Maintain the oncotic pressure). ‫اذا صار أي مشكلة فيهم بصير في‬
edema.
The other functions: carriers, enzymes, antibodies, hormones.

*causes of increase plasma protein concentration:

1.dehdration(because of vomiting or diarrhea ): concentration of fluid so
concentration plasma protein so Hyperproteinemia. (‫)كل البروتينات بتزيد بنفس الكمية‬
2. multiple myeloma: increase in specific type: gamma globulin.
3.high protein diet.

causes of decrease plasma protein concentration:

1.problems in the liver.
2. malnutrition.
3. nephrotic syndrome,it is kidney damage in glomerular basement membrane this affect on
reabsorption and filtration to protein that’s lead to excessive protein in the urine. (clinically these
Patients have edema in soft tissue: periorbital, testicular)
4.sever Burns: decrease to hydrostatic pressure because of inflammation and expansion in
Capillaries.

*what is the method or principle they used in laboratory to know if the solution have
peptide bond (have protein) or not:
soluble proteins + copper salt (in alkaline solution NaOH) purple color complex
(called biuret solution)
The intensity of the color is directly proportion to the concentration of the protein in the
solution.

*plasma glucose level:

What is the important organ that controls the blood glucose level in the
circulation?? pancreas
*Pancreas has 2 functions:
1. 99%exocrine function of pancreas: release of pancreatic lipases enzymes to the
second portion of the duodenum.
2. 1% endocrine function of pancreas endocrine: is mediated by islets of
Langerhans (abundant in tail of pancreas).
*islets of Langerhans contain these cells:
1. B-cells: secretion and production of insulin hormone (prevent excessive
elevation of blood glucose level by helping glucose to enter the cells)
2. alpha cells: secretion of glucagon (one of the counter regulatory hormone,
decrease glucose in the tissue)
3. Delta cells: secrets major inhibitory hormone to all hormones in the body:
somatostatin
4. F-cells or PP cells: secrets pancreatic polypeptide.
*what are the causes of increase blood glucose level (hyperglycemia)?
1.autoimmune diseases: do destruction to beta cell dysfunction ( in Diabetes type
1)
2.resistance to insulin (Diabetes type 2).
3.over activity of thyroid gland, pituitary gland and adrenal gland.

*what are the causes of decrease blood glucose level (hypoglycemia)?

1.insulin overdose.
2.insulin secreting tumor(insulinoma): tumor in beta cells lead to excessive
production to insulin so there is always low glucose in circulation.
3.Addison disease: Autoimmune disease affects the Adrenal gland (this gland
secrets glucocorticoids (counter regulatory hormone))

**Tests for blood glucose level:

1. Fasting Blood sugar: off calories to more than 8 hours.
Normal range: <100 mg/dl
Impaired fasting state: 100-125mg/dl (at high risk they will be diabetes
mellitus)
Diabetic: >= 126 mg/dl

2.Random blood glucose level: (‫)مش صايم‬

Normal range: <140 mg/dl
Increase risk to diabetes: 140-199 mg/dl
Diabetic: >= 200mg/dl
3.Hemoglobin A1c:reflex degree of storage glucose in the last three
months)‫اكم‬ ‫ر‬
‫(الب ي‬
Normal: <5.7%
Increase risk to diabetes : 5.7-6.4%
Diabetic: >6.5%
4. is The most sensitive test 75 g oral glucose tolerances test (OGTT)
Give the patient 75g glucose ,after 2 hours I will check the sugar values.
Values are the same as Random blood glucose level.

‫مسلمان‬
‫ي‬ ‫ضىح‬