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TAN

HEMOGLOBIN
TANA
TIGLAO
TIMBOL
UMALI
Group 9
1A2
LEARNING OBJECTIVES
At the end of the session, students must be able to discuss the:
1. Hemoglobin synthesis
a. Heme synthesis
b. Underlying genetics for globin synthesis
2. Molecular basis of the following clinical conditions
a. Iron deficiency anemia
b. Sickle cell anemia
c. Thalassemia
3. Synthesis and importance of HbA1c in the management of diabetes
mellitus.
HEMOGLOBIN (Hb, Hgb)

- A tetramer composed of two identical globin chains


- each of which binds a heme molecule
- Hemoglobin is the primary constituent of red blood cell cytoplasm and
transports molecular oxygen from the lungs to the tissues
- Normal values:
- Male: 12.7 to 18.3 g/dL
- Female: 11.0 to 15.8 g/dL
HEMOGLOBIN SYNTHESIS
HEME

GLOBIN
HEME
- Pigmented iron-containing nonprotein part of hemoglobin
- Four heme groups in a hemoglobin molecule, each containing one
ferrous ion in the center.
- Oxygen binds the ferrous ion and is transported from an area of high
to low oxygen concentration.
GLOBIN
- Protein constituent of hemoglobin
- Two identical pairs of globin chains bind four heme molecules to form
hemoglobin
- Forms the tetrameric molecule or the globin portion of the hemoglobin
- Each globin chain is covalently attached to a heme moiety
- globular or ellipsoid
HEME SYNTHESIS

Photo retrieved from: https://www.slideshare.net/ashokktt/heme-biosynthesis-and-its-disorders-porphyria


MITOCHONDRIA

Glycine

d- ALA Synthase d- aminolevulinic acid

Succinyl CoA
d- aminolevulinic acid d-ALA dehydratase Porphobilinogen
uroporphyrinogen I
Porphobilinogen synthase
Uroporphyrinogen I
uroporphyrinogen III
Uroporphyrinogen I synthase
Uroporphyrinogen III
Uroporphyrinogen uroporphyrinogen Coproporphyrinogen
III decarboxylase III
MITOCHONDRIA

Coproporphyrinogen coproporphyrinogen Protoporphyrinogen


III oxidase IX
MITOCHONDRIA

Protoporphyrinogen Protoporphyrinogen
Protoporphyrin XI
IX oxidase
MITOCHONDRIA

Protoporphyrin XI Ferrochelatase Heme


GLOBIN BIOSYNTHESIS
● Globin genes involved: a, b, d, e, g, z
○ Alpha
○ Beta
○ Delta
○ Epsilon
○ Gamma
○ Zeta
● Short arm of chromosome 16: controls the a & z chain
● Short arm of chromosome 11: controls the b, d, e, & g chains
GLOBIN BIOSYNTHESIS
● The 4 porphyrin heme moieties lie in 4
regularly spaced clefts on the tetramer
surface
● Globin portion of a normal hemoglobin
contains:
○ 2 chains of chromosome 16
○ 2 chains of chromosome 11
Photo retrieved from: Rodak’s Hematology: Clinical Principles and Practice, 5th Edition by Keohane, Elaine (2016). Elsevier Inc: Saunders
Photo retrieved from: Rodak’s Hematology: Clinical Principles and Practice, 5th Edition by Keohane, Elaine (2016). Elsevier Inc:
Saunders
Globin release from
ribosomes

Each globin chain


binds to a heme
molecule

Heterodimer
2 heterodimers
combine

Tetramer

Hemoglobin molecule
Photo retrieved from: Rodak’s Hematology: Clinical Principles and Practice, 5th Edition by Keohane, Elaine (2016). Elsevier Inc: Saunders
Carbohydrate Metabolism: Glucose Determination
Stanbio Glucose Liquicolor (Oxidase) Procedure No. 1070

Intended use: Quantitative Determination of Glucose in serum, plasma and CSF

Principle:

Source: Noninvasive Method for Glucose Level Estimation from Journal of Diabetes and Metabolism
Carbohydrate Metabolism: Glucose Determination
PROCEDURE

Blood sample = Serum


Incubation = 37°C
RBS Experiment Results
Glucose Conc.
(mg/dL)

Absorbance
RBS Experiment Results
Glucose Conc.
(mg/dL)

Absorbance
Carbohydrate Metabolism: Glucose Determination
Expected values:

Normal range:

● Serum/Plasma: 70-105 mg/dL (3.89-5.83 mmol/L)


● CSF: 40-75 mg/dL (2.22-4.17 mmol/L)
NOTE: The ranges should serve only as guidelines. It is ultimately the responsibility of the laboratory to establish its own
range of expected values, since differences exist between instruments, laboratories and local populations.

Computation:
Au = Absorbance of the unknown
Glucose (mg/dL) = Au / As x concentration of the std. As = Absorbance of the standard = 0.280
Concentration of the standard = 100
Carbohydrate Metabolism: Glucose Determination
● Important in the diagnosis and management of hyperglycemia and
hypoglycemia

A. Hyperglycemia - Diabetes Mellitus, severe stress


B. Hypoglycemia - result of an insulinoma, insulin administration, inborn error of
carbohydrate metabolism or fasting
Quantitative Estimation of Hemoglobin: Cyanmethemoglobin Method

Principle:
Potassium ferricyanide Potassium cyanide

Hemoglobin (Fe 2+) Methemoglobin (Fe 3+) Cyanmethemoglobin


Oxidation

Cyanmethemoglobin reagent:

● 0.75 mM Potassium cyanide


● 0.6 mM Potassium ferricyanide
● Surfactant - increases RBC lysis and decreases turbidity caused by abnormal proteins
Quantitative Estimation of Hemoglobin: Cyanmethemoglobin Method

PROCEDURE:

Blood sample = Whole Blood


Incubation = 37°C
Hemoglobin Experiment Results
Hgb Conc.
(g/dL)

Absorbance
Hemoglobin Experiment Results
Hgb Conc.
(g/dL)

Absorbance
Quantitative Estimation of Hemoglobin: Cyanmethemoglobin Method

Expected values:

Normal range:

● Male: 12.7-18.3 g/dL


● Female: 11.0-15.8 g/dL
NOTE: The ranges should serve only as guidelines. It is ultimately the responsibility of the laboratory to establish its own
range of expected values, since differences exist between instruments, laboratories and local populations.

Computation: Au = Absorbance of the unknown


As = Absorbance of the standard = 0.542
Hemoglobin (g/dL) = Au / As x concentration of the std. Concentration of the standard = 20
CLINICAL CORRELATIONS
1. Iron deficiency anemia
2. Sickle cell anemia
3. Thalassemia
4. Hemoglobin of patients with diabetes mellitus
IRON DEFICIENCY ANEMIA
- common type of anemia
- blood has a lower than normal number of red blood cells
- develops over time if your body doesn't have enough iron to build healthy
red blood cells
- without enough iron, your body starts using the iron it has stored.
- soon, the stored iron gets used up. Insufficient storage of iron will make fewer red
blood cells also, making haemoglobin less normal
- Microcytic, hypochromic anemia.
- Characterized by pallor, fatigue and weakness.
- Often caused by low dietary iron intake or chronic blood loss.
SICKLE CELL ANEMIA
● disorder of the blood caused by an
inherited abnormal hemoglobin
● autosomal recessive genetic disorder
● characterized by an
abnormally-shaped (sickle-shaped)
red blood cell
● atypical hemoglobin S(SS)

Photo retrieved from:


https://geneed.nlm.nih.gov/topic_subtopic.php?tid=142&sid=149 on
September 30, 2018
SICKLE CELL ANEMIA SIGNS AND SYMPTOMS
● fatigue and anemia
● shortness of breath
● fatigue
● episodes of pain
● painful swelling of hands and
feet
● frequent infections
● delayed growth
Photo retrieved from:
● vision problems
https://www.microscopyu.com/gallery-images/sickle-cell-anemia-at-40x-magnification
● jaundice
Photo retrieved from:
http://mazurekopticalservices.co.uk/using-optical-microscope-detect-disease/ on
September 30, 2018.
SICKLE CELL ANEMIA
Hemoglobin S - is defined by the structural formula :

6Glu-->Val
α 2β 2
● β chain at position 6, Glutamic acid (-1) is replaced by valine(0).
● Glutamic acid is polar, the β chain extends outward from the surface to bind to
water.
● Valine is non polar also extends outward but instead of binding to water it
seeks to
○ a hydrophobic niche to bind with.
SICKLE CELL ANEMIA

● When Hb S is fully oxygenated, the quaternary structure of the molecule does


not produce a hydrophobic pocket for valine to bind to and this maintains its
biconcave shape like of a normal RBC.
● But when Hb S is deoxygenated this creates a hydrophobic pocket for valine
to bind with specifically in the area of phenylalanine 85 and leucine 88.
Photo retrieved from: Rodak’s Hematology: Clinical Principles and Practice, 5th Edition by Keohane, Elaine
(2016). Elsevier Inc: Saunders
Photo retrieved from: Rodak’s Hematology: Clinical Principles and Practice, 5th Edition by Keohane, Elaine (2016).
Elsevier Inc: Saunders
THALASSEMIA

● inherited blood disorder


● hemoglobin production is abnormal
● may have mild or severe anemia
TYPES OF THALASSEMIA
● classified based on two things
1. specific part of hemoglobin not synthesized (alpha or beta)
2. severity (minor or major)
ALPHA THALASSEMIA
- an inherited deletion of chromosome-16
- reduced or absent synthesis of α-globin chains
- 4 genes involved
- you may get 2 from each parent

➔ 1 mutated gene ➔ 3 mutated genes


◆ no signs/symptoms of ◆ symptoms will be moderate
thalassemia; but a carrier, to severe
may be passed to children
➔ 2 mutated genes ➔ 4 mutated genes
◆ mild symptoms; alpha ◆ rare; affected fetuses may
thalassemia have severe anemia and are
usually stillborn
TYPES OF ALPHA THALASSEMIA
Hemoglobin H (HbH disease) Hemoglobin Barts
(Alpha Thalassemia Major)
● Milder form of alpha thalassemia ● Fetus with 4 alpha globin deletions.
● One normal α-globin gene and tetramers ● Characterized by hydrops fetalis, which is a
of b-globin (HbH). condition of having excess fluid that builds
● absence of three alpha globin genes. up before birth.
● can cause jaundice, ● Symptoms include severe anemia,
hepatosplenomegaly, and mild to hepatosplenomegaly, heart defects, and
moderate anemia. abnormality in the urinary system or
genitalia.
BETA THALASSEMIA
- reduced or absent synthesis of β-globin chains
- 2 genes involved, 1 from each parent

➔ 1 mutated gene
◆ mild signs and symptoms
◆ thalassemia minor
➔ 2 mutated genes
◆ Moderate to severe
◆ Thalassemia major
◆ babies are usually born
healthy but deveop signs
and symptoms within the first
two years of life
BETA THALASSEMIA
Major/Homozygous Minor/Heterozygous
● aka Cooley’s anemia ● One beta chain is normal
● markedly decreased rate of synthesis or ● HgbA is slightly decreased
absence of both beta chains resulting in an ○ Compensation: HgbA2 is
excess of alpha chains slightly increased.
● no HgbA is produced ● Laboratory findings:
● usually so severe that it requires regular ○ Mild microcytic, hypochromic
blood transfusions anemia
● Laboratory findings:
○ Severe microcytic, hypochromic anemia
○ Increased serum iron and bilirubin
THALASSEMIA
Common symptoms Severe symptoms

Fatigue bone deformities


Weakness - expanded bone marrow, wide bones
Pale or yellowish skin enlarged spleen (splenomegaly)
Facial bone deformities - since there are more damaged blood
Slow growth cells, the spleen works harder than
normal
Abdominal swelling
slowed growth rates
Dark urine
- may cause delay in puberty
heart problems
- congestive heart failure, arrhythmias
HEMOGLOBIN OF PATIENTS WITH DIABETES MELLITUS

● Diabetes mellitus
○ group of metabolic disorders in
which there are high blood sugar
levels over a prolonged period
CONDITIONS OF PATIENTS WITH DIABETES MELLITUS

● Type I diabetes ● Gestational diabetes


○ due to AUTOIMMUNE b-cell mellitus (GDM)
destruction → insulin ○ diabetes in 2nd or 3rd trimester of
deficiency pregnancy
● Type II diabetes ● Monogenic diabetes
○ due to PROGRESSIVE LOSS syndromes
of b-cell insulin secretion → ○ neonatal diabetes, maturity-onset
insulin resistance diabetes of the young (MODY),
pancreatitis, cystic fibrosis
HEMOGLOBIN OF PATIENTS WITH DIABETES MELLITUS
Hemoglobin
Tests for diabetes
➔ makes the blood red
➔ 90% of hemoglobin is Hgb A 1. Fasting plasma glucose (FPG)
2. 2-h plasma glucose (2-h PG)
Hemoglobin A during 75-g oral glucose
➔ Minor components include tolerance test (OGTT)
◆ A1c (Hemoglobin A1c) 3. A1c test
◆ A2b
◆ A1a1
◆ A1a2
HbA1c (Hemoglobin A1c)
➔ minor component of hemoglobin A that is being measured to check the
average plasma glucose concentration in the body

➔ a GLYCOSYLATED HEMOGLOBIN
◆ Glucose + Hemoglobin = Glycosylated hemoglobin
◆ where glucose is bound

➔ made when glucose in the body sticks to the hemoglobin of the RBC
◆ there is an elevated amount of HbA1c in the blood in patients with diabetes
mellitus
HbA1c synthesis
➔ results from a non-enzymatic
attachment of a hexose molecule
(glucose) to the N-terminal amino
acid (valine) of the hemoglobin
molecule
➔ attachment of the glucose molecule
happens continuously over the
lifespan of the erythrocyte
◆ dependent on:
● blood glucose concentration
● duration of erythrocyte
exposure to blood glucose

(Baloyo, Miranda, Barriero, et al., 2015)


(Baloyo, Miranda, Barriero, et al., 2015)
HEMOGLOBIN OF PATIENTS WITH DIABETES MELLITUS
In a normal individual:

● Normal glucose level: normal amount of glycosylated hemoglobin (HbA1c)

NOTE: as plasma glucose increases, glycated hemoglobin increases

What happens when an individual has high levels of glucose in the body?

● Higher levels of glucose in body:


○ glucose will greatly attach to hemoglobin → higher chances of having more HbA1c
concentration than normal

NOTE: once a hemoglobin molecule is glycated, it is non-reversible


HEMOGLOBIN OF PATIENTS WITH DIABETES MELLITUS
A higher HbA1c is an indicator that your blood sugar is increasing and
therefore, an HbA1c test provides a useful test or indicator to see if a
person is diabetic, pre-diabetic, or still within the normal range

HbA1c test

● Measures the percentage of HbA1c in your hemoglobin


● Checks the individual if he/she is diabetic, pre-diabetic, or normal
● An AVERAGE test of the blood sugar level over the course of three
months
● Tests how much glucose is bound to the hemoglobin or how much
glycated hemoglobin (HbA1c) is in the blood
https://www.webmd.com/diabetes/guide/gly
cated-hemoglobin-test-hba1c
HEMOGLOBIN OF PATIENTS WITH DIABETES MELLITUS
Why is the HbA1c test an AVERAGE test of the blood sugar over the course of three
months?

● HbA1c test indicates the amount of sugar you have in your body for this time
period due to replenishment and loss of RBCs daily

NOTE: blood glucose test is different from HbA1c since the former measures blood
glucose in the very moment whilst HbA1c is an average measurement

HbA1c test indicates how well the diabetes has been controlled over the past
months and this test could be used to adjust the medications of a diabetic patient
depending on the concentration of HbA1c in the blood
HEMOGLOBIN OF PATIENTS WITH DIABETES MELLITUS
To summarize:

● Glucose + Hemoglobin = GLYCATED/GLYCOSYLATED HEMOGLOBIN (HbA1c)

● The HIGHER the blood glucose, the HIGHER the HbA1c concentration
○ Often seen in diabetic patients

● Normal range of HbA1c concentration: <5.7%


● High range of HbA2c concentration: >6.5%
● HbA1c test: measures blood glucose content or HbA1c concentration over a
long period of time (LONG TERM)
SOURCES
Alpha thalassemia - Genetics Home Reference - NIH. (n.d.). Retrieved October 2018, from
https://ghr.nlm.nih.gov/condition/alpha-thalassemia

Interactive, W. (n.d.). Pathology Thread. Retrieved October 2018, from


https://www.med-ed.virginia.edu/courses/path/innes/nh/globin.cfm

Jr, W. C. (n.d.). Sickle Cell Anemia: Learn About SCD Symptoms and Treatment. Retrieved October 2018, from
https://www.medicinenet.com/sickle_cell/article.htm#how_is_sickle_cell_anemia_inherited

Keohane, E. M., Walenga, J. M., Smith, L. J., & Rodak, B. F. (2016). Rodaks hematology: Clinical principles and
applications. St. Louis, MO: Elsevier/Saunders.

Sickle cell disease - Genetics Home Reference - NIH. (n.d.). Retrieved October 2018, from
https://ghr.nlm.nih.gov/condition/sickle-cell-disease

Thalassemia: MedlinePlus Medical Encyclopedia. (n.d.). Retrieved October 2018, from


https://medlineplus.gov/ency/article/000587.htm

Thalassemia. (2018, August 28). Retrieved October, 2018, from https://www.cdc.gov/ncbddd/thalassemia/facts.html

Thalassemia. (2016, November 02). Retrieved October 7, 2018, from


https://www.mayoclinic.org/diseases-conditions/thalassemia/symptoms-causes/syc-20354995
SOURCES

American Diabetes Association. (2018, January 01). Classification and Diagnosis of Diabetes: Standards of
Medical Care in Diabetes-2018. Retrieved October 06, 2018, from
http://care.diabetesjournals.org/content/41/Supplement_1/S13

Health Direct (Ed.). (n.d.). HbA1c test. Retrieved October 06, 2018, from
https://www.healthdirect.gov.au/hba1c-test

Dansinger, M. (2016, October 26). Hemoglobin A1c (HbA1c) Test for Diabetes. Retrieved October 06, 2018,
from https://www.webmd.com/diabetes/guide/glycated-hemoglobin-test-hba1c

Random Glucose Tests: Testing Stability from


https://www.healthline.com/health/type-2-diabetes/random-glucose-testing