MLS 11A | LECTURE

CLINICAL CHEMISTRY
MIDTERM: S.Y. 2022-2023

CONTENTS
● Carbohydrates
● Lipids and Lipoproteins
● Amino Acids

REFERENCES
➔ Lecture Notes and Side Notes (Discussion)
● Bishop et al. Clinical Chemistry: Principles, Techniques, and
Correlations 8th ed.
➢ Rodriguez, M.T. (2018). Clinical Chemistry Review Handbook for MT
→ Carbohydrates contain the aldose (aldehyde) group or the
ketose (ketone) group
CARBOHYDRATES
2. CHO can form glycosidic bonds with other CHO &
➔ Carbohydrates are compounds containing C=O and OH
other substances
functional groups. It has a general formula of Cx(H2O)y
→ Compounds that contain carbon, hydrogen, and oxygen
→ C=O and -OH are very important in the structure of
carbohydrates

Functions of Carbohydrates:
1. Major source of energy in the body
2. Serves as major entry point to the metabolic pathway
3. Provides structural integrity to cell membrane
4. Determines the antigenicity of molecules
- Blood types are identified because of the component of
the membrane of RBCs, which is the carbohydrate
➔ Carbohydrates are the immediate sources of energy.
Organisms rely on the oxidation of complex compounds to
obtain energy. Although other compounds could be sources
of this energy, carbohydrates are the primary source for
brain, erythrocytes, and retinal cells in humans.
➔ Exogenous carbohydrates are usually obtained from plant
sources. The endogenous carbohydrates are in the form of
“animal starch” or glycogen. When body carbohydrates are
excess, they are stored in the liver and muscles as glycogen.
Muscle glycogen, however, is available only for the muscles
and not for other tissues. Compared to plant starch, animal
starch is not a good source of carbohydrates because
glycogen disintegrates upon death of an animal.
Chemical Properties of CHO:
1. Some CHO have reducing property
- This is the property of CHO which is utilized in the labs
for the measurement of CHO
- Reducing CHO means it has a ketone or aldehyde
group
- Example: Glucose, maltose, fructose, galactose,
and lactose
- If the bond forms with carbon on CHO other than the
anomeric carbon, the anomeric carbon (functional group)
is unaltered and the resulting compound remains a
reducing substance
→ The anomeric carbon is found in carbon 1 of the carbohydrate
structure
- If the bond is formed with the anomeric carbon on the
other CHO, the resulting compound is not a reducing
compound anymore
- Non-reducing sugars do not have active aldose or
ketose groups, therefore, can not oxidize or reduce other
compounds (Ex. sucrose)
- All monosaccharides and many disaccharides [maltose
& lactose] are reducing agents
Sources of Carbohydrates:
1. Endogenous → inside the body (muscles)
2. Exogenous → from the plants and food sources
Why are meats NOT good sources for carbohydrates?
- When animals die or are slaughtered, carbohydrates in
the muscles easily disintegrate

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Carbohydrates are Classified According to the 4 Properties: ➔ Double ➔ Sucrose (glucose

1. Size of the base carbon chain. Ex. Trios contains 3 structures of + fructose)
carbon atoms; pentose contains 5 carbon atoms, hexose Oligosaccharide/Dis monosaccharide ➔ Maltose (glucose
contains 6 carbon atoms accharide + glucose)
2. Location of the CO function group. Ex. Aldose form ➔ Lactose (glucose
which has an aldehyde as its functional group; ketose + galactose)
form which has ketone as the functional group. The ➔ Polymers of ➔ Starch
carbon atom in the functional group is called anomeric Polysaccharide monosaccharide ➔ Cellulose
carbon ➔ Most abundant ➔ Glycogen
Glycosidic bond links a monosaccharide to another
monosaccharide

→ Carbohydrates are also classified according to sources:

CHO Sources
Sucrose ➔ Cane sugar, beet sugar,
molasses
Lactose ➔ Milk and milk products
Glucose ➔ Fruits, honey, corn syrup
Fructose ➔ Fruits and honey
3. Number of sugar units. Ex. Monosaccharides,
disaccharides, polysaccharides. Monosaccharides are the 4. Stereochemistry. Stereoisomers have the same order
building blocks of carbohydrates. They include glucose, and types of bonds, but different spatial arrangements
fructose, and Galactose. Disaccharides are made up of 2 and different properties. For each asymmetric carbon,
monosaccharide units (sucrose= glucose + fructose; there are 2n possible isomers; therefore, there are 21 , or
maltose= glucose + glucose; lactose= glucose + 2 forms of glyceraldehyde. These isomers are mirror
Galactose). Polysaccharides are polymers of image.
monosaccharides. Examples are starch, cellulose, and
glycogen. Linkage is by glycoside bonds.

➔ There are several models used to represent carbohydrates.

These are: Fisher projection, which has the aldehyde or
ketone at the top of the drawing. The carbons are numbered
starting at the aldehyde or ketone end. The compound can
Type Description Examples be represented as straight chain or might be linked to show
➔ Building blocks ➔ Glucose a representation of the cyclic, hemiacetal form Haworth
of CHO ➔ Fructose projection represents the compound in the cyclic form that is
Monosaccharide ➔ Contains 3-8 ➔ Galactose more representative of the actual structure. This structure is
carbons formed when the functional group reacts with an alcohol
➔ Contains only 1 group on the same sugar to form a ring called a hemiacetal
aldehyde or ring.
ketone group

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2 MODELS USED TO REPRESENT CARBOHYDRATES

Fisher Projection
➔ Has the aldehyde or ketone ➔ Represents the compound
group at the top of the
drawing
➔ Carbons are numbered ➔ Structure is formed when
starting at the aldehyde or
ketone end
➔ Compound can be
represented as straight
chain or might be linked to
show a representation of
the cyclic
Haworth Projection
in the cyclic form
the functional group reacts
with an alcohol group on
the same sugar to form a
ring called a hemiacetal
ring
What Would Happen to Carbohydrates Taken into the Body
➔ They are converted to glycogen if not utilized. If utilized as
fuel (converted to ATP), it produces carbon dioxide and
water as products of metabolism.
➔ They are used in biological synthesis of other types of
compounds found in the body such as fatty acids and some
amino acids
➔ They are involved in producing other biological
substances like glycolipids, glycoproteins, heparin, nucleic
acids & other substances

Glucose Metabolism
➔ Carbohydrate digestion starts in the mouth where the
enzyme amylase is secreted by the salivary glands. The
enzyme converts starch into starch dextrins, maltose and
glucose. The parietal cells of the stomach produces HCl that
CHO Digestion
maintains a certain pH in the area. It is with this reason that
➔ Begins in the mouth
when the food fragments reach the stomach, amylase
➔ Acted by salivary amylase (PTYALIN)
activity is stopped. Carbohydrate digestion is continued only
➔ Cellulose is not changed & it contributes to the bulk of stool
when the chyme from the stomach reaches the duodenum
in the colon
of the small intestine. In the intestine, the bicarbonate-rich
➔ Broken down to monosaccharides (Once the amylase and
secretion of the pancreas neutralizes the acidity of the
other enzymes would act on CHO)
chyme. It is also in this phase of digestion that the pancreas
→ pancreatic amylase is also called AMYLOPSIN
secretes amylase that continues the digestion process. The
➔ Absorbed in intestinal cells
end products of this process include maltose and
isomaltose. These products with the other disaccharides

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ingested are then acted upon by disaccharidases located in source of energy for many vital organs including the brain
the brush border of the cells lining the lumen of the small and the heart in cases of starvation. Further, the utilization
intestine. Cellulose fibers are not changed as they pass by of fatty acids as source of energy spares the proteins from
the gastrointestinal tract because enzymes do not act on further proteolysis which could result to muscle mass
them. This reason explains the importance of cellulose in reduction.
the bulk of the stool in the colon and in the normal passage
of wastes. NOTE
➔ There are instances that brush border enzymes are ➔ Glycolysis: metabolism of glucose molecule to pyruvate or
genetically absent or are destroyed. An example of this lactate for production of energy
condition is lactase deficiency that results to lactase ➔ Gluconeogenesis: formation of glucose 6-phosphate from
intolerance. Lactose can not be degraded and it stays in the non-carbohydrate sources
intestine where it is acted upon by lactose-fermenting ➔ Glycogenolysis: breakdown of glycogen to glucose for use
bacteria. This further results in the formation of gases as energy
leading to abdominal cramps and flatulence. ➔ Glycogenesis: conversion of glucose to glycogen for
➔ Monosaccharides are absorbed by the intestines. Once storage
monosaccharides reach the liver via the portal circulation, ➔ Lipogenesis: Conversion of carbohydrates to fatty acids
interconversion of hexoses occurs. This process ensures the ➔ Lipolysis: decomposition of fats
conversion of fructose and galactose into glucose. The
conversion of Galactose to glucose is catalyzed by
galactokinase and Galactose-1-phosphate uridyl
transferase. The absence of these enzymes results in the
leakage of Galactose in the circulation, a condition known as
galactosemia. A clinical consequence of this condition is the
accumulation of a by-product called galactilol. Galactilol
contributes to cataract formation.
➔ After the interconversion process, the liver dispatches the
glucose molecules to the different extrahepatic tissues in
need of an energy source. Glucose enters into the glycolytic
pathway or Embden-Meyerhof pathway, which forms
energy from glucose in the form of adenosine triphosphate
(ATP). This is the process of glycolysis. On the other side, if
glucose is not needed, it is stored in the liver in the form of
glycogen. The process of building up glycogen from glucose
is called glycogenesis.
➔ In the event when tissues are depleted with glucose, for
example in the case of starvation, glycogen is converted
back to glucose in a process called glycogenolysis. The
reserve, however, is only about 100 g that will last only
about 10-18 hours fasting. Thus, to maintain the blood
glucose level within normal range, the muscle proteins must
be mobilized. The proteins are degraded into amino acids,
many of which are glucogenic in nature. These glucogenic
amino acids are converted to glucose in the liver in the
process called gluconeogenesis.
➔ While this protein break down occurs, a degradation of
triglycerides in the adipose tissues also happens. The
products are fatty acids. These fatty acids are brought to the
liver where they are converted to acetyl CoA, the precursor
of ketone bodies. Ketone bodies serve as an important

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➔ Glycolysis is the catabolism of glucose to pyruvate or

lactate for ATP production (Embden-Meyerhof Pathway and
Krebs Cycle)
- Produces a gross amount of 38 ATPs minus 2 ATPs
METABOLIC PROCESSES
spent during the process, thereby producing 1 for every
➔ Glycogenesis is the process of glycogen formation by
1 molecule of glucose, in net proceeds of 36 ATPs.
enzyme action on glucose [glycogenesis → meaning,
Glycolysis will be producing plenty of ATPs
“formation of glycogen”]

➔ Glycogenolysis is the breakdown of glycogen (with ➔ Gluconeogenesis is the formation of glucose from amino
formation of G-6-Phosphate that is used for energy acids and lipids
production); reverse of glycogenesis - Formation of glucose from non-carbohydrate sources
- The liver and the kidneys can perform glycogenolysis
that, with which, the glucose (end product) can be used
by the body especially in times of fasting
- The muscles could only convert its stored glycogen back
into glucose, which can only be utilized in the muscle
cells and cannot be shared in other parts of the body
due to the lack of G-6-Phosphate

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However, due to an increased elimination of insulin, it is
seen that the ratio of C-peptide to insulin becomes 5:1. It is
important to note that although it is insulin which is the
active hormone, C-peptide plays a significant role in
differentiating exogenous from endogenous insulin.
C-peptide has become a marker for the endogenous
production of insulin. A high level of C-peptide (> 1.9
ng/mL) suggests hyperinsulism which is characterized by
severe hypoglycemia.
➔ A 55-amino acid polypeptide released when plasma
glucose is greater than 5 mmol/L via the glucose
transporter 2
➔ Released by the ß-cell of pancreatic islets of langerhans

→ Proteins and lipids are the sources of carbohydrates in

gluconeogenesis
→ When the reaction is reversed, there will be the formation of
fats (lipogenesis) and proteins. When these lipids and proteins
enter the acetyl CoA (which is a common pathway to the citric
acid cycle), these would eventually be used and converted into
ATPs
→ In glycolysis, glucose is being processed and enter the Krebs
cycle, eventually producing ATPs
→ Glycogenolysis involves the formation of glucose from
glycogen
→ Glycogenesis is the formation of glycogen from glucose

Regulation of Carbohydrate Metabolism

➔ The reference range for fasting glucose varies from one
laboratory to another. In general, blood glucose after an 8
hour fasting is between 70 to 110 mg/dL (3.9- 6.1 mmol/L).
The normal range of blood glucose is controlled by several
organs of the body like the liver, pancreas and other
endocrine glands

1. Insulin
➔ Insulin is the primary hormone responsible for the entry of
glucose into the cell. Of all the hormones involved in
→ Insulin comes from preproinsulin, eventually undergoing
glucose homeostasis, it is only insulin that lowers blood
proteolysis, forming proinsulin, which would be cleaved to form
glucose. It can be referred to as a hypoglycemic agent.
the mature insulin
➔ Insulin is synthesized by the ß-cells of islets of Langerhans
Note that this process could occur only if the insulin is
in the pancreas as a pro-hormone called pro-insulin.
endogenously produced (insulin is produced by beta cells, and
Pro-insulin is processed by cleavage to form Cpeptide and
this process would not occur if insulin comes from
insulin. There is a 1:1 molar ratio of C-peptide and insulin
medications/injections)

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C-peptide
➔ C-peptide is the cleaved part during the proteolysis of
proinsulin to mature insulin. C-peptide can only be observed
if the insulin is produced from the body. C-peptide cannot
be observed if it is an injectable insulin
➔ Cleaved from proinsulin
➔ Measured in conjunction with insulin and blood glucose to
help identify the cause of hypoglycemia.
➔ C-peptide and insulin are secreted in equimolar amounts in
the portal vein, but the ratio in serum is about 5:1 to 15:1.
C-peptide is greater than the amount of insulin.
➔ The molar concentration of C-peptide in blood is higher
than that of insulin due to hepatic clearance of insulin.
Supposedly the ratio is 1:1, however because of the
degradation of the insulin, the ratio becomes 5:1 to 15:1.

→ The presence of insulin would change some configurations of

the receptor cells, allowing the entry of glucose into the cells,
where this glucose can be processed (i.e., undergoing glycolysis).
➔ Insulin binds to specific cell receptors, enhancing glucose
entry to muscle and adipose tissues
➔ Insulin-induced activation of enzymes stimulates glucose
incorporation into glycogen in liver and muscles

OTHER USES OF C-PEPTIDE MEASUREMENT

➔ To follow-up evaluations after pancreatectomy and after
pancreatic transplant.
➔ C-peptide and proinsulin are primarily degraded by the
kidneys. Elevated levels are seen in renal failure.

RELATIONSHIP OF C-PEPTIDE AND INSULIN LEVELS IN

DIFFERENT HYPOGLYCEMIC STATES

Conditions Insulin C-Peptide

Insulinoma Increased Increased

Insulin Treatment Increased Decreased

Type 1 Diabetes Absolute Decreased

deficiency

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SUMMARY OF ACTION OF INSULIN IN LIVER, MUSCLE, AND FACTORS THAT STIMULATE INSULIN RELEASE
ADIPOSE TISSUES: 1. Glucose. Increase of glucose intake signals the pancreas
1. Effects on Carbohydrates. In the liver, insulin inhibits to secrete insulin. The release of insulin causes an
gluconeogenesis and glycogenolysis. In the muscles and increased movement of glucose into the cells and
liver, insulin increases glycogenesis. The removal of increased glucose metabolism. Insulin is normally
glucose from the blood is made through stimulating the released when glucose levels are high and is not
relocation of the insulin-sensitive GLUT-4 glucose released when glucose levels are decreased.
transported from the cytoplasm to cell membranes of 2. Amino acids. After a protein-rich meal, insulin is
adipose and skeletal muscles. In the muscles and increased. Arginine stimulates the ß-cells of pancreas to
adipose tissue, insulin increases glucose uptake. The secrete insulin.
ability in the glucose uptake has been attributed to the 3. Gastrointestinal Hormones. Incretins ( like
ability of insulin to bind to its receptors on the cells’ glucagon-like peptide-1& glucose-dependent
membrane. insulinotropic peptide) stimulate insulin secretion after
2. Effects on Lipids. Insulin decreases triglyceride ingestion of food. These hormones cause an increased
breakdown (lipolysis) in the adipose tissue by inhibiting level of insulin before blood glucose elevates.
the hormone-sensitive lipase. It promotes triglyceride - Secretin stimulates insulin secretion after ingestion of
synthesis by increasing transport of glucose into the food
adipocytes providing glycerol-3-phosphate which is
needed in triglyceride synthesis. Further, insulin also ➔ The next set of hormones has an opposing effect to insulin.
increases the activity of the lipoprotein lipase or lipemia They elevate blood glucose level.
clearing factor in the plasma. This provides the needed
fatty acid in the synthesis of triglycerides. 2. Glucagon
3. Effects on Protein. Insulin stimulates the entry of amino ➔ Glucagon is the primary
acids into the cells. It promotes the synthesis of proteins hormone responsible for
in most tissues. increasing glucose levels.
It is produced by the α-
cells of the islets of
Langerhans in the
pancreas. It is released
during stress and fasting
states. It acts by
increasing plasma
glucose levels by
glycogenolysis, and
lipolysis in the liver and
an increase in
gluconeogenesis. Unlike
insulin, glucagon targets only the liver. It is called the
“hyperglycemic hormone”.
➔ A single polypeptide synthesized by α- cells of the
→ Items in red would lead to the increase in blood sugar; the
pancreatic islets
green ones which pertain to the action of insulin act to regulate
→ it opposes the action of insulin
blood sugar
➔ In longer term, insulin regulates growth and development,
and the expression of certain genes

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Note: If insulin will bring up your glucose, glucagon will bring
down glucose.
→ During fasting or starvation, it is glucagon which is being
produced by the pancreas
Effects of glucagon:
1. Liver: Increasing glycogenolysis and gluconeogenesis
- Decreased glycogenesis
- Increased hepatocellular survival and lipid oxidation
2. Intestine: Decreasing gut motility
3. Kidney: Increasing rate of GFR and water
reabsorption
4. Adipose tissue: increasing lipolysis
5. Brain: increasing satiety and glucose production
6. Heart: increasing cardiac inotropic effect, glucose
oxidation and increasing glycolysis
FACTORS THAT STIMULATE GLUCAGON RELEASE
1. low blood glucose- an overnight fast will result to an
elevated glucagon levels to prevent hypoglycemia.
2. amino acids- amino acids stimulate insulin and
glucagon to prevent hypoglycemia
3. epinephrine- this together with norepinephrine
stimulate the release of glucagon. During stress, trauma,
and excessive exercise, regardless of the glucose
concentration in the blood, glucagon levels are elevated
in anticipation of an increased glucose use. In contrast,
insulin level is low.
EFFECTS OF GLUCAGON ON:
1. carbohydrate metabolism: glucagon leads to an
immediate increase in blood sugar. This is due to the
breakdown of liver glycogen and an increased in
gluconeogenesis.
2. lipid metabolism: glucagon favors hepatic oxidation of
fatty acids and subsequent formation of ketone bodies
from acetyl CoA.
3. protein metabolism: Glucagon increases the uptake of
amino acids by the liver resulting in availability for
carbon skeletons needed for gluconeogenesis. As a
consequence, plasma amino acids are decreased.
3. Epinephrine
➔ Epinephrine is a hormone of the adrenal medulla.
➔ It is known as the “fight or flight” hormone.
➔ It activates the adenylate cyclase which produces cyclic
adenosine monophosphate (cAMP).
➔ High levels of cAMP activate the enzyme phosphorylase
causing increased glycogenolysis.
→ Since there is glycogenolysis (conversion of glycogen to
glucose), this means that the blood glucose level would be
elevated
➔ It also stimulates the breakdown of triglyceride by
activating the hormone-sensitive lipase of adipocytes.
➔ Hence, epinephrine promotes glycogenolysis and lipolysis,
inhibits insulin secretion, and inhibits the
insulin-dependent uptake of glucose by the peripheral
tissues.
4. Cortisol
➔ Cortisol is a hormone produced by the adrenal cortex.
➔ It mainly promotes gluconeogenesis (production of energy
from noncarbohydrate sources) by promoting protein
catabolism and deamination.
➔ It inhibits glucose metabolism in the peripheral tissues.
➔ Cortisol and growth hormone are less important in the
short-term maintenance of blood glucose concentration
because their role is for the long term regulation of glucose
metabolism
5. Growth Hormone
➔ The antagonistic action of insulin is achieved by inhibiting
the uptake of glucose by cells and lipogenesis from
carbohydrates.
➔ It also promotes release of fatty acids from adipocytes.
➔ GIANTS- have high GH which means they are also prone to
hyperglycemia
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6. ACTH HORMONES AFFECTING GLUCOSE LEVELS
➔ ACTH stimulates the adrenal cortex to release cortisol and Hormone Effect
increases plasma glucose levels by converting liver ➔ Hypoglycemic (brings
glycogen to glucose (glycogenolysis) and by promoting Insulin down glucose to a lower
gluconeogenesis. level)
→ ACTH is produced by the anterior pituitary gland Glucagon
Epinephrine
7. Thyroid Hormones Growth hormone & ➔ Hyperglycemic
➔ Thyroid hormones promote Adrenocorticotropic hormone
absorption of glucose in the Thyroid hormones
intestinal tract. They also stimulate Glucocorticoids
glycogenolysis and accelerates the
degradation of insulin.
➔ T3 and T4 increase glycogenolysis,
gluconeogenesis, & intestinal
absorption of glucose

8. Somatostatin
➔ Somatostatin is produced by
the D cells (delta cells) of the
pancreas.
➔ It inhibits pituitary,
gastrointestinal, and
pancreatic hormones.
➔ Somatostatin inhibits insulin, → The pancreas has several cells - some of which are involved in
glucagon, and growth the regulation of glucose.
hormone release 1. Alpha cells secrete glucagon
2. Beta cells secrete insulin
3. Delta cells secrete somatostatin

ROLE OF LIVER
➔ Liver is the most important organ maintaining a constant
glucose supply for other tissues, including the brain
→ This is primarily the reason why even if you have not yet
eaten, your brain continuously receives energy
→ In cases that there is already a limited supply of glucose, then
glucose will be obtained from other non-carbohydrate sources
(such as fats and proteins, which will be used up to provide
energy for the brain)
➔ Liver is also important in controlling postprandial plasma
glucose concentrations
➔ The entry of glucose into liver and cerebral cells is NOT
directly affected by insulin, but depends on the ECF
(extracellular fluid) glucose concentration

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(≥11.1 mmol/L)

CLINICAL CORRELATIONS
➔ Hypoglycemia: FBS is below 45 mg/dL
➔ Hyperglycemia: FBS is above 100 mg/dL
➔ Glucosuria: renal threshold for glucose exceeds 160-180
mg/dL
→ Glucosuria is the presence of glucose in the urine. Take note
that normally, we do not excrete glucose in the urine. If the
plasma carries glucose, this will be reabsorbed in the body;
however, if the plasma has elevated glucose levels that
exceeds the renal threshold (160-180 mg/dL), then glucose
can pass through the glomerular filtration membrane and will
be excreted in the urine
➔ DM: Disorder of glucose metabolism

ROLE OF RENAL CORTEX Pathology in Carbohydrate Metabolism

➔ The renal cortex is the only other tissue capable of
gluconeogenesis and converting G6P to glucose Hypoglycemia
➔ The gluconeogenic capacity of kidneys help in H+ ion ➔ Hypoglycemia : is a decreased glucose level resulting in
homeostasis and during prolonged fasting symptoms associated with the nervous system functions. It
can result from several causes- transient and insignificant
Categories of FBS (Fasting Blood Sugar) but other causes can be life threatening. Hypoglycemia can
→ Fasting: no eating overnight for at least 8 hours-10 hours, or deprive fuel to the brain that results to coma or brain death.
maximum of 12 hours (w/ lipid profile); do not reach 16 hours ➔ At about 45 mg/dL glucose, warning signs and symptoms
because a lot of changes would already occur at this particular occur
time. → This could be transient and relatively insignificant at times;
however, this can also be life-threatening
Normal Fasting 70-99 mg/dL
Glucose (3.9-5.5 mmol/L)
Impaired Fasting 100-125 mg/dL
Glucose (value exceeds (5.6-6.9 mmol/L)
the normal range, but
cannot yet be classified
as DM)
Provisional Diabetes ≥ 126 mg/dL
Diagnosis (7 mmol/L)

Categories of Oral Glucose Tolerance

Normal Glucose 2-H PG ≤ 140 mg/dL

Tolerance (≤ 7.8 mmol/L)
Impaired Glucose 2-H PG 140-199 mg/dL
Tolerance (value (7.8-11.1 mmol/L) SYMPTOMS OF HYPOGLYCEMIA (WHIPPLE’S TRIAD) ARE
exceeds the normal ➔ hypoglycemia attacks is precipitated by fasting
range, but cannot yet be ➔ plasma glucose level is <45 or <50 mg/dL (2.75 mmol/L)
classified as DM) ➔ symptoms are relieved by administration of glucose
Provisional Diabetes 2-H PG ≥ 200 mg/dL

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BODY’S DEFENSE IN HYPOGLYCEMIA equal to 70 mg/dL be used as cut-off and alert level to
➔ Glucagon release prevent hypoglycemic episode.
➔ Insulin inhibited b. Fasting hypoglycemia (Postabsorptive) is rare but
➔ Epinephrine release serious. It is also known as postabsorptive
◆ Hormones that would help elevate blood hypoglycemia. The symptoms are that of
glucose will be increased, and insulin would be neuroglycopenic type. Low blood glucose is due to
inhibited. insulinoma (tumor in beta cells), adrenal insufficiency, in
fasting individual who have consumed large quantities
of ethanol ( ethanol leads to accumulation of NADH
which diverts the intermediates of glucose into other
pathways resulting in decreased hepatic production of
glucose) or in increased rate of glucose utilization as a
result of elevated insulin. The patient may lose
consciousness, experience convulsion and coma if left
untreated

REASONS OF FASTING HYPOGLYCEMIA

➔ Hepatocellular damage (damage to the liver)
➔ Adrenal insufficiency
➔ In fasting individual who have consumed large quantities of
ethanol
➔ Increased rate of glucose utilization as a result of elevated
insulin.

DIVISIONS OF HYPOGLYCEMIC SYMPTOMS Remember C peptide:

➔ Adrenergic symptoms are mediated by epinephrine release
as regulated by the hypothalamus in response to
hypoglycemia. These symptoms are anxiety, weakness,
palpitation, tremor, and sweating. These symptoms occur
when glucose blood levels fall abruptly
➔ Neuroglycopenic symptoms are due to impaired glucose
delivery to the brain. They are headache, confusion, slurred
speech, seizures, coma, and death. They occur from gradual
decline in glucose ( below 40 mg/dL) that deprives the CNS
of fuel but fails to trigger an epinephrine release.
NOTE: epinephrine → increases blood glucose Proinsulin → cleaved → mature insulin; what was left behind is
the C peptide. Note that we can only observe C peptide if the
CLASSIFICATION OF HYPOGLYCEMIA insulin is produced by the body. We could not have C peptide
a. Postprandial hypoglycemia (Reactive) is common. This production if it is an injectable insulin
is known as reactive hypoglycemia. Described only the
timing of hypoglycemia but not the etiology. An C peptide
exaggerated insulin release occurs after a meal ( about 4 ➔ Cleaved from proinsulin
hours after) causing transient hypoglycemia with mild ➔ Measured in conjunction with insulin and blood glucose to
adrenergic symptoms. The plasma glucose returns to help identify cause of hypoglycemia
normal even without feeding the patient. Treatment is ➔ C-peptide and insulin are secreted in equimolar amounts in
requiring the patient to eat frequent small meals than the portal vein, but the ratio in serum is about 5:1 to 15:1
the usual 3 big meals. ADA suggests that for individuals → C-peptide is greater than the amount of insulin
with DM, plasma glucose concentration of less than or

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MIDTERM: S.Y. 2022-2023
➔ The molar concentration of C-peptide in blood is higher level of sugar because the baby used to have an elevation of
than that of insulin due to hepatic clearance of insulin glucose while the baby is inside the womb. Once the
→ Supposedly, the ratio should be 1:1; however, because of the umbilical cord is cut, there is also a sudden stop of high
degradation of insulin glucose supply to the baby. That is why babies born from
GDM mothers who are not managed well could experience
Other uses of C peptide measurement: hypoglycemia.
➔ To follow-up evaluations after pancreatectomy and after
pancreatic transplant Genetic Defects in CHO Metabolism
➔ C-peptide and proinsulin are primarily degraded by the ➔ Glycogen storage disease results from deficiency of specific
kidneys. Elevated levels are seen in renal failure enzymes
→ The kidneys are also degrading the C peptide ➔ The most common is glucose-6-phosphatase deficiency
type 1 (von Gierke disease)
RELATIONSHIP OF C-PEPTIDE AND INSULIN LEVELS IN
DIFFERENT HYPOGLYCEMIC STATES von Gierke disease
Conditions Insulin C-Peptide ➔ Severe hypoglycemia that coincides with metabolic acidosis,
Insulinoma Increased Increased ketonemia, elevated lactate and alanine, hyperlipidemia,
Insulin Treatment Increased Decreased uricemia, growth retardation
Type I Diabetes Absolute deficiency Decreased ➔ Glycogen ≠ glucose = hypoglycemia
→ There is no way that the increase in glycogen could elevate
CHEMISTRY FINDINGS OF HYPOGLYCEMIA glucose levels
Analyte Result ➔ Glycogen builds in liver = hepatomegaly (enlargement of
liver cells)
Glucose ≤ 2.5 mmol/L
➔ Glycogen accumulation is irreversible but disease can be
Insulin ≥ 6 μU/mL
kept under control by avoiding hypoglycemia
C-peptide ≥ 0.2 nmol/L
Proinsulin ≥ 5 pmol/L
Other enzyme defects causing hypoglycemia:
ß-hydroxybuterate ≥ 2.7 mmol/L
➔ Glycogen synthase
→ Ketones will also be elevated, primarily because there is
➔ Fructose-1,6-biphosphatase
hypoglycemia and the body tries to compensate by using
➔ Phosphoenolpyruvate carboxykinase
noncarbohydrate sources for energy
➔ Pyruvate carboxylase

How to diagnose hyperinsulinism or insulinomas (pancreatic

Galactosemia
ß-cell tumor)
➔ Cause of failure to thrive syndrome in infants
➔ RIA (Radioimmunoassay)
➔ A congenital deficiency of one of 3 enzymes involved in
➔ stimulatory tests ( insulinomas show that insulin is still
galactose metabolism, resulting in increased levels in
present in the blood even after 72 hours fasting)
galactose in plasma
➔ C-peptide ( normal C-peptide is < 1.2 ng/mL)
➔ Common deficient enzyme is galactose-1-phosphate
→ C-peptide is also used during follow-up evaluations
uridyltransferase
after pancreatectomy/ transplant as well as in diagnosis
➔ Can cause mental retardation & cataracts
of Renal Failure (if >15)
Hyperglycemia
In newborn, hypoglycemia is determined when glucose in
➔ Hyperglycemia: is an increase in plasma glucose levels. It is
➔ Preterm/low birth infants during the first week: <25 mg/dL
caused by an imbalance of hormones. In a healthy
➔ Full term infants <35 mg/dL (Born after 9 months)
individual, this triggers the release of insulin which
➔ Birth to the first 72 hours: <45 mg/dL
increases the permeability of cell membrane in the liver,
How is it possible for newborns to have hypoglycemia?
muscle, and adipose tissues.
➔ When the mother is having Gestational Diabetes mellitus
(GDM) and the baby is born. It is possible that the baby,
upon cutting the umbilical cord, will experience a very low

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HYPERGLYCEMIA MAY BE DUE TO: Random blood ≥ 200 mg/dL (≥11.1
➔ IV infusion of glucose-containing fluids (which was not glucose mmol/L)
properly monitored) Plus symptoms of
➔ Severe stress such as trauma, MI, or cerebrovascular DM
accidents ➔ ADA (American Diabetes Association) recommends that all
➔ Imbalance of hormones. adults older than age 45 years should have be tested
glucose using HbA1c, FBS or 2-hour 75g OGTT every 3
LABORATORY FINDINGS IN HYPERGLYCEMIA years unless the individual is diagnosed with diabetes
1. increased glucose in plasma and urine ➔ Glucose tests should be done at an earlier age and more
2. increased urine specific gravity frequently in individuals who display:
3. increased serum and urine osmolality ◆ Habitually physically inactive
4. ketones in serum and urine (ketonemia and ketonuria) ◆ Obesity (120% of desirable body weight or
5. decreased blood and urine pH (acidosis). body mass index (BMI) of 27 kg/mm2)
→ There is the accumulation of hydrogen ions and the ketone ◆ Family history of diabetes in a first-degree
bodies that leads to ketoacidosis. relative
6. electrolyte imbalance ◆ Membership in a high-risk minority
population
SYMPTOMS OF HYPERGLYCEMIA ◆ History of GDM or delivering a baby > 9lb
➔ Increased thirst ◆ Hypertension (>149/90 mmHg)
➔ Headaches ◆ Elevated TAG levels (>250 mg/dL)
➔ Difficulty concentrating ◆ Low HDL concentration (<35 mg/dL)
➔ Blurred vision ◆ AIC ≥5.7%, IGT or IFG on previous testing
➔ Frequent urination ◆ A history of impaired fasting
➔ Fatigue (weak, tired feeling) glucose/impaired glucose tolerance
➔ Weight lose ◆ Women with polycystic ovarian syndrome
(PCOS)
➔ Hyperglycemia is one of the signs of diabetes. Diabetes
◆ Other clinical conditions associated with
mellitus is a disorder of glucose metabolism that results in
insulin resistance (ex. acanthosis nigricans)
insulin deficiency and lack of carbohydrate tolerance.
◆ History of cardiovascular disease
➔ There is an increasing incidence of adolescent type 2 DM in
Diabetes Mellitus
the past years
➔ Diabetes Mellitus (DM): is a group of metabolic disease
➔ These children are overweight (BMI >85th percentile for
characterized by hyperglycemia resulting from defects in
age and sex, weight for height >85 percentile or weight
insulin excretion, insulin action, or both. An elevation of
>120% of ideal for height) plus have any two of the
plasma glucose affects vital organs including the eyes (
following risk factors:
blindness), kidneys( renal failure), and the heart ( heart
◆ Family history of diabetes 2 in first-or
attack and strokes). Amputation is a consequence of
second-degree relative
microangiopathy
◆ Race
➔ A disorder of glucose metabolism that results in insulin
◆ Maternal history of diabetes or GDM
deficiency and lack of carbohydrate tolerance
◆ Signs of insulin resistance or conditions
associated with insulin resistance (e.g.
WHO DIAGNOSTIC CRITERIA FOR DM
acanthosis nigricans, hypertension,
Test Values for Impaired Values indicating dyslipidemia, PCOS)
Criterion DM
HbA1c ≥ 6.5% SCREENING FOR THESE CHILDREN:
Fasting blood 110-125 mg/dL ≥ 126 mg/dL (≥ 7 ➔ Testing at the age of 10 years or at the onset of puberty
glucose mmol/L) ➔ If puberty occurs at a younger age, follow glucose test
2-hour postprandial 140-199 mg/dL ≥ 200 mg/dL (≥11.1 every 2 years
blood glucose mmol/L)

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ADA/WHO CATEGORIES OF DM
➔ Type 1
➔ Type 2
➔ GDM
➔ MODY
➔ Associated with secondary factors [genetic defects of beta
cell, pancreatic disease, endocrine disease, drug/chemical
induced, insulin receptor abnormality]
SYMPTOMS OF DIABETES
Symptoms of DM Description
Frequent urination as a result of osmotic
ability of glucose. As the filtered blood
Polyuria passes through the renal tubules, more
water is absorbed by the hyperosmotic.
Ultrafiltrate thus increasing the urinary
output
Polydipsia An excessive thirst as a consequence of
increased urine output
An excessive hunger. However, even
Polyphagia with a high glucose, peripheral tissues
have lost their ability to take up glucose,
resulting to deprivation of glucose
➔ The 1979 classification of DM was changed by the
International Expert Committee on the Diagnosis and
Classification of DM in 1995. The changes included
eliminating the older terms of IDDM (insulin dependent DM)
and NIDDM (non-insulin dependent insulin DM). The
categories type 1 and 2 were retained with the use of Arabic
numerals instead of Roman numerals. Now, the ADA/WHO
guidelines recommend the following categories of diabetes:
TYPES OF DIABETES
Category Description
➔ Formerly called IDDM
➔ There is inappropriate
hyperglycemia
➔ Often childhood and juvenile onset
➔ ß-cell destruction
Type 1 diabetes ➔ Absolute insulin deficiency
➔ Thin patients
➔ Ketoacidosis
➔ Constitutes only 10% to 20% of all
cases of DM
➔ Usually initiated by environmental
factors or infection(virus) with
genetic predisposition.
➔ Type 1 DM autoantibody marker
◆ NOTE: TYPE 1 DM is an
autoimmune disease
◆ autoantibodies: islet cell
autoantibodies;
◆ insulin autoantibodies,
◆ glutamic acid decarboxylase
autoantibodies;
◆ tyrosine phosphatase IA-2 and
IA2B autoantibodies
➔ Formerly called NIDDM
➔ A multifactorial disease
➔ Hyperglycemia is due to Insulin
resistance or decreased insulin
receptors on cell surfaces
➔ Insulin resistance with an insulin
secretory defect
Type 2 diabetes ➔ Relative insulin deficiency
→ The body could still produce insulin.
The problem is that the insulin receptors
are not responding to your insulin.
➔ Rarely to have ketoacidosis
➔ Patients are more likely to go into
hyperosmolar coma and of
increased risk of developing macro
and microvascular complications
➔ Associated with secondary
conditions
➔ Genetic defects of ß-cell function
Other specific types of ➔ Pancreatic disease
diabetes ➔ Endocrine disease
➔ Drug or chemical induced
➔ Insulin receptor abnormalities
➔ Other genetic syndromes
➔ Glucose intolerance during
pregnancy
→ This becomes very common because
of the tests done also among pregnant
patients or women
Gestational diabetes ➔ Due to metabolic and hormonal
mellitus (GDM) changes
➔ Blood glucose returns to normal
postpartum (after giving birth)
➔ But there could be increased
perinatal complications and
increased risk of DM in later life.
MODY(Maturity Onset ➔ Rare form that is inherited in an
Diabetes melitus of the autosomal dominant fashion
Young)

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Type 1 Diabetes Mellitus
➔ Type 1 diabetes mellitus is characterized by inappropriate
hyperglycemia primarily due to cellular-mediated
autoimmune destruction of the pancreatic ß-cell, causing an
absolute deficiency of insulin secretion. The destruction of
ß-cell is initiated by an environmental factor or infection (by
a viral) in individuals with a genetic predisposition. As a
result, the ß-cell identifies itself as “non-self”. With this
reason that T cell infiltrates the islets causing an
inflammation called insulinitis”. The autoimmune attack
results to depletion of ß-cells. It takes 80-90% of the
ß-cells to be destroyed before DM symptoms occur. This
type of DM occurs commonly in childhood and adolescence.
◆ Type 1 DM is genetically related and → When you have type I DM, there is low/No insulin; your
constitutes only 10-20% of all diabetes but is immune system would attack or destroy the beta cells. There is
life threatening because the patient is prone to ketoacidosis or the breakdown of fats and protein and there is
ketoacidosis. Patients with this type of DM also inability to use the glucose due to low insulin. Remember, Insulin
manifest hypertriglyceridemia which is a result facilitates the entrance of glucose into the cells. Because of this,
of the depression of the activity of the glucose in the blood would increase.
lipoprotein lipase. LPL requires insulin to
function

ASIDE FROM THE TRIAD SYMPTOMS SEEN, THERE ARE

➔ Rapid weight loss
➔ Hyperventilation
➔ Mental confusion
➔ Loss of consciousness ( due to a high glucose in the brain)

Idiopathic Type 1 Diabetes Mellitus

➔ Idiopathic type 1 DM is a type with no known etiology.
➔ It is strongly inherited but does not have ß-cell
autoimmunity.
➔ Have episodic insulin replacement.

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Ketoacidosis (DKA = Diabetic Ketoacidosis)
➔ Ketoacidosis (DKA=diabetic ketoacidosis) may present
feature of type 1 DM or may develop in patient who omits
insulin treatment. This is very rare in type 2DM
Type 2 Diabetes Mellitus
➔ Type 2 diabetes mellitus is characterized by hyperglycemia
due to an individual’s resistance to insulin with an insulin
secretory effect. This resistance results in a relative
deficiency in insulin. This is a milder form than type 1 with
ketoacidosis occurring very rarely. However, patients are
more likely to go into a hyper-osmolar coma and of
increased risk of developing macro and microvascular
complications.
➔ Type 2 affects older people. It makes up the major diabetes
cases, although often goes undiagnosed for many years.
→ However, with several factors that go along with elevation of
blood sugar, this can be identified.
➔ Patients are generally obese and have an increased
percentage of body fat distribution in the abdominal region.
➔ Type 2 DM is strongly associated with:
◆ genetic predisposition
◆ increase in age,
◆ obesity, and
◆ lack of physical exercise.
→ If there is insulin resistance to your receptor, there would be
increased glucose production and insufficient glucose disposal,
leading to the accumulation of glucose in the system.
Other Specific Types of Diabetes Mellitus
➔ Other specific types of DM are associated with secondary
conditions, including genetic defects of β-cell function or
insulin action, pancreatic disease, diseases of endocrine in
origin, drug-or chemical-induced insulin receptor
abnormalities.The characteristics and prognosis of this type
depends on the primary disorder. MODY or maturity-onset
diabetes of youth is a rare form of diabetes that is inherited
in an autosomal dominant fashion. Diagnosis of MODY is
made to young people who develop type 2 diabetes or have
a very strong history of diabetes.
Gestational Diabetes Mellitus
➔ Gestational DM (GDM) is any degree of glucose intolerance
with onset or first recognition during pregnancy. Patients
normally return to normal after giving birth. However, GDM
is associated with increased perinatal complications and an
increased risk for development of diabetes in later years.
Also, infants are at increased risk for respiratory distress
syndrome, hypokalemia, and hyperbilirubinemia. Insulin
production is stimulated in Infants of diabetic mothers. This
is the explanation why they suffer to hypoglycemia after the
umbilical cord is severed because of the abrupt termination
of an oversupply of glucose.
→ Since the baby has been used to a very elevated glucose, and
when the umbilical cord is cut, there is a high possibility that the
baby will have hypoglycemia. Because the baby is used to having
high glucose levels, it causes the baby to gain weight (“big baby”.
BABIES BORN TO DM MOTHERS HAVE INCREASED RISK OF:
➔ Respiratory distress
➔ Hypocalcemia
➔ Hyperbilirubinemia
➔ Hypoglycemia
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PATHOPHYSIOLOGY OF DM Galactosemia
Findings Type 1 Type 2 ➔ Cause of failure to thrive syndrome in infants
Hyperglycemia Present Present ➔ A congenital deficiency of one of 3 enzymes involved in
Glucosuria (if more Present Present galactose metabolism, resulting in increased galactose
than 180mg/dl) levels in plasma
Ketone Formation High Rare, but greater ➔ Common deficient enzyme is galactose-1-phosphate
tendency to develop uridyltransferase
hyerosmolal ➔ Can cause mental retardation and cataracts.
nonketotic states
Pancreatic High glucagon Normal or Pathophysiology of DM
Hormones Absence insulin hyperinsulinemia ➔ Both type 1 and 2 DM are characterized by
Metabolic process Common is Oxidation Fatty acid hyperglycemia and glucosuria. Glucosuria occurs when
gluconeogenesis and is inhibited. Fatty the renal tubular transporter system for glucose
lipolysis acids incorporated becomes saturated as plasma glucose concentration
into TAG and exceeds 180 mg/dL.
released as LDL. ➔ Type 1 DM has a highest tendency to produce ketones
Type 2 seldom generate ketone bodies but has a greater
Genetic defects in carbohydrate metabolism tendency to develop hyperosmolar non-ketotic states.
a. Glycogen storage disease = results from enzyme The difference in glucagon and insulin concentration of
deficiency the 2 types is responsible for the production of ketone
➔ Glucose-6-phosphatase deficiency type 1 (most bodies through increased ß-oxidation. In type 1 there is
common type) the absence of insulin with an increase of glucagon. This
◆ also called von Gierke disease permits gluconeogenesis and lipolysis to occur. In type 2
◆ an autosomal recessive disease insulin is present, thus, glucagon is attenuated. In type 2,
◆ characterized by (severe) hypoglycemia with fatty oxidation is inhibited, thus, fatty acids are
metabolic acidosis, ketonemia, elevated lactate incorporated into triglycerides for release as very
and alanine, hyperlipidemia, uricemia, and low-density lipoprotein.
growth retardation.
◆ glycogen cannot be converted back to glucose= Pathophysiology of DM
hypoglycemia = can be corrected by liver Findings Type 1 Type 2
transplantation Hyperglycemia Present Present
◆ there is glycogen build-up in the liver = Glucosuria (If more Present Present
hepatomegaly (enlargement of liver cells) than 180 mg/dL)
◆ glycogen accumulation is irreversible but Ketone Formation High Rare, but greater
disease can be kept under control by avoiding tendency to develop
hypoglycemia. hyperosmolar
nonketotic states
➔ Other enzymes when deficient cause hypoglycemia : Pancreatic hormones High Glucagon Normal or
glycogen synthase, fructose-1,6-biphosphatase, Absence insulin hyperinsulinemia
phosphoenolpyruvate carboxykinase, & pyruvate Metabolic Processes Common is Oxidation Fatty acid
carboxylase. gluconeogenesis and is inhibited. Fatty
➔ Debrancher enzyme deficiency does not cause lipolysis acids incorporated
hypoglycemia but hepatomegaly. into TAG and
Alimentary hypoglycemia = hypoglycemia occurs due to increase released as LDL
reels of insulin in response to rapid absorption of nutrients after a
meal or the rapid secretion of insulin-releasing gastric factors ➔ Due to acidosis, bicarbonate and total carbon dioxide are
usually decreased due to Kussmaul-Kein respiration
(deep respiration). This is a compensatory mechanism

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to blow off carbon dioxide and remove hydrogen ions.
This is primarily the reason why there is acidosis in
diabetes melitus (particularly Type I DM).
➔ Serum osmolality is high due to hyperglycemia; sodium
is low (due to polyuria (increased urine output, together
with the increased excretion of electrolytes such as
sodium) and in part due to a shift of water from cells
because of hyperglycemia). Grossly elevated
triglycerides displace plasma volume and give the
appearance of low electrolytes
In Type II DM
➔ In type 2, there is an imbalance between production and
elimination of glucose in the urine. Often this state is
precipitated by heart disease, stroke or pancreatitis.
➔ There is also severe dehydration that contributes to the
inability to excrete glucose in the urine.
➔ The severe hyperosmolar state inhibits the ability of
glucagons to stimulate lipolysis.
➔ The gross elevation in glucose and osmolality,
elevation in BUN(blood-urea-nitrogen), and the
absence of ketones are the laboratory findings that
distinguish type 2 from type 1 diabetes.
Complication of Diabetes Mellitus
➔ End-stage renal disease (ERD)
➔ Nontraumatic amputation
➔ Blindness in adults aging 20 to 74 years old
➔ Diabetic neuropathy (nerve damage) in 60% to 70% of
diabetic patients
➔ Atherosclerotic disease
➔ 2 to 4 times predisposition to heart and cardiovascular
diseases
➔ Because of the insufficiency of insulin and glucagon excess,
it will lead to decreased cellular uptake of glucose,
increased protein catabolism, and increased lipolysis.
Eventually, when not managed properly or the patient won't
go to a physician for treatment, will lead to dehydration and
acidosis, leading into coma or death.
Diagnosing Diabetes Mellitus
➔ ADA recommends that all adults older than age 45 years
should have be tested glucose using HbA1c, FBS or 2-hr 75
g OGTT every 3 years unless the individual is diagnosed
with diabetes. Glucose tests should be done at an earlier
age and more frequently in individuals who display:
◆ Habitually physically inactive
◆ Obesity ( 120% of desirable body weight or
body mass index (BMI) of 27 kg/mm2)
◆ Family history of diabetes in a first-degree
relative
◆ Membership in a high-risk minority population
◆ History of GDM or delivering a baby >9lb
◆ Hypertension (>140/90 mmHg)
● Low HDL concentration (<35 mg/dL)
◆ Elevated TAG levels (>250 mg/dL)
◆ AIC ≥5.7%, IGT or IFG on previous testing
◆ A history of impaired fasting glucose/impaired
glucose tolerance
◆ Women with polycystic ovarian syndrome
(POCS)
◆ Other clinical conditions associated with insulin
resistance ( ex. acanthosis nigricans)
◆ History of cardiovascular disease
➔ There is an increasing incidence of adolescent type 2
DM in the past years. These children are overweight (
BMI >85th percentile for age and sex, weight for height
>85th percentile or weight >120% of ideal for height)
plus have any two of the following risk factors:
◆ Family history of diabetes 2 in first-or
second-degree relative
◆ Race
◆ Signs of insulin resistance or conditions
associated with insulin resistance ( e.g.
◆ acanthosis nigricans, hypertension,
dyslipidemia, PCOS)
◆ Maternal history of diabetes or GDM
● Must be closely monitored and be
subjected to early screening of DM
➔ Screening for these children:
◆ Testing at the age of 10 years or at the onset of
puberty
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◆ If puberty occurs at a younger age, follow
glucose test every 2 years
Laboratory Diagnosis for Diabetes Mellitus
➔ Specimen requirement
- Use of whole blood or plasma. In most cases we make
use of serum or plasma.
- Separate cellular elements from serum or plasma.
- Leukocytosis – high wbc, esp. if there is bacterial
contamination.
- Glucose in the blood is a food to the bacteria.
◆ Glucose metabolism occurs in unprocessed
specimens in the following conditions.
● At room temperature the rate of
metabolism is 7mg/dL/hr
● Storage at 4°C will cause a loss of
approximate 2mg/dL/hr
● In bacterial contamination and
leukocytosis (increased WBC), the
rate of metabolism is higher.
Organisms in the cells would need
glucose, which leads to a falsely
decreased glucose level upon
examination.
● Whole blood specimens tend to give
decreased glucose reading compared
to plasma by 10% to 15%.
Percentage difference may vary with
the hematocrit of the patient. The
hematocrit of the patient should be
taken into consideration.
● When refrigerated, 2 mg of sodium
fluoride/mL (gray top tube) (could be
added) of whole blood prevents
glycolysis for up to 48 hours.
Glucose measurement
➔ Glucose measurement can be done using serum, plasma,
urine, CSF, or whole blood. The glucose concentration in
whole blood is approximately 11% lower than plasma.
The reason for this is: The glucose concentration in the
water that makes up plasma is equal to that of
erythrocytes. Plasma has greater water content than
erythrocytes and, therefore, exhibits higher glucose
levels than whole blood.
➔ Serum/plasma must be refrigerated and separated from
cells within 1 hour
➔ There are characteristics of carbohydrates that are useful
for their detection and quantification in the body fluids
a. ability of glucose to function as a reducing agent.
➔ Glucose converts cupric ions ( deep blue color) in
alkaline solution to cuprous ions (red precipitate).
➔ Examples if these solution: Benedict’s ( stabilizer: citrate)
and Fehling’s (stabilizer: tartrate) reagents
b. Ability of glucose to form Schiff bases with aromatic
amines
➔ - O-toluidine in a hot acidic solution will yield a colored
compound
➔ at absorbance maximum 630 nm.
➔ - Galactose (aldohexose) and mannose (aldopentose)
react and
➔ interfere with the reaction
➔ - This method is replaced by the following enzymatic
methods:
Glucose oxidase
➔ This reaction is inhibited by high concentrations of uric
acid, vitamin C, bilirubin, glutathione, creatinine,
L-cysteine, L-dopa, dopamine, methyldopa, and citric
acid.
➔ Glucose oxidase is the most specific enzyme reacting
with only β-D-glucose converting it to gluconic acid.
Oxygen is consumed and H2O2 is produced.
➔ First reaction is monitored by measuring the rate of
disappearance of oxygen by using oxygen electrode or
by consuming H2O2
➔ Horseradish peroxidase catalyzes the second reaction.
Peroxidase interferences: increased uric acid, bilirubin,
ascorbic acid can cause falsely decreased value. Bleach
can result to falsely elevated value.
➔ Polarographic technique can provide direct measurement
of O2, avoiding the above interferences.
➔ The shift in absorbance in the second reaction can be
measured photometrically
➔ First and second reactions ( couple reaction) is called
Trinder reaction
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Trinder Reaction (Glucose Oxidase Reaction)
➔ Glucose+O2+H2O —--->Gluconic acid+H2O2
Glucose oxidase
➔ H2O2+reduced chromogen—--> oxidized
chromogen+H2O (Peroxidase)
➔ H2O2 must be eliminated to prevent the reaction from
reversing by use of catalase and molybdate.
Hexokinase method
➔ The generally accepted method for measuring glucose.
➔ More accurate than glucose oxidase because the
coupling reaction using G-6-PD is highly specific
➔ Reference method, not affected by uric acid and ascorbic
acid
➔ Gross hemolysis and elevated bilirubin = falsely
decreased value
➔ NaF - significant anticoagulant;
➔ The glucose enzymatic procedure cannot be carried out if
the specimen is collected using fluoride because it
deactivates or inactivates the enzyme. Enzymatic activity
for glucose can only be done if the specimen collected is
in a plain tube or other tubes but not the sodium
fluoride.
➔ Do not subject specimen to enzyme method if NaF is
used
Tests to detect and monitor DM
➔ Fasting Blood Sugar (FBS) or Random Blood Glucose
(FBG)
◆ FBS is commonly used to screen diabetes
mellitus. The patient is instructed not to take
anything (except water) for 8-10 hours ( not
longer than 16 hours) overnight. FBG has a
diurnal variation with a mean FBG higher in the
morning than in the afternoon. Missed
diagnosis for DM patients occurs if the FBS is
done in the afternoon.
TABLE 4: ADA diagnostic criteria for FBG
Normal 70-99 mg/dl (3.9 to 5.5 mmol/L)
impaired 100 mg/dl to 125 mg/dl (5.6 to 6.9
mmol/L)
diabetes 126 mg/dl or higher (≥7 mmol/L or
higher)
➔ Random Blood Sugar (RBS) or Random Blood glucose
(RBG)- also called casual glucose testing
◆ The highest rise in blood glucose level after a
meal is 10-15 mg/dL. Normally, glucose blood
levels of individuals below 65 years old is
within 45-130 mg/dL (2.5-7.2 mmol/L). In older
patients RBS is 180 mg/dL is still considered
normal.
➔ Two-Hour Post-Prandial Glucose (2-hr PP)
→ “Challenge test”
◆ This is a valuable screening tool for detecting
diabetes mellitus since the maximum glucose
level is usually reached 60-90 minutes after a
meal. This is commonly used to screen possible
Diabetes mellitus. The test is performed when
the patient demonstrates symptoms of DM or
when results of FBS suggest diabetes. The 2-hr
PP is usually a component of the glucose
tolerance test.
◆ Before the test, the patient is instructed to eat a
balanced meal or one containing 75 g of
carbohydrates 2 hours before the test
◆ He is not allowed to smoke and do strenuous
exercises after the meal
→ nicotine stimulates Epinephrine production
which increases glucose levels
◆ Patient is given a solution of 75 g of glucose 2
hours before the test. He is not allowed to
smoke and do strenuous exercises after the
meal. Result is interpreted as:
TABLE 5: ADA diagnostic criteria for oral glucose tolerance
Normal glucose 2-h <140 mg/dL (≤7.8 mmol/L
tolerance
Impaired glucose 2-h 140 to 199 mg/dL (7.8-11.1
tolerance mmol/L)
Provisional diabetes 2-h ≥200 mg/dL (≥11.1 mmol/L)
diagnosis
An issue however, arises on the reference level of 2 hr PP values
according to age. Test values fluctuate according to the patient’s
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age after 50 years, normal levels could be as high as 160 mg/dL.
In a younger patient a glucose value of more than 140 suggests
incipient diabetes and needs further evaluation.
Diagnostic Criteria for GDM
➔ Glucose Tolerance Test (GTT)
◆ This is used to diagnose GDM. The
International Association of Diabetes and
Pregnancy Study groups recommend that all
nondiabetic pregnant women should be
screened for GDM at 24 to 28 weeks of
gestation.
◆ GTT can either be oral GTT or by the
intravenous GTT. The oral GGT can be one- or
two-step approach.
Oral glucose tolerance test
➔ Most sensitive for evaluating borderline cases of DM
➔ Commonly performed to screen GDM (gestational
diabetes) in the 3rd trimester of pregnancy
➔ Can be one or two-step approach
Oral glucose tolerance test one-step approach:
1. FBS is first tested.
2. The patient is given a 75 g (pink liquid) glucose load (
glucola or glucoplus). Glucola is one of the examples of
the glucose preparation. Today, there is a lot of
preparation that is used for this test.
3. Blood is taken after 1 hour and 2 hours
4. 1h plasma glucose <140 mg/dL = no GDM
5. 1h plasma glucose more than 140 mg/dL = GDM
possible, proceed to the 3h GTT
6. Glucose measurements done after 1 hour and 2 hours.
- In a one-step approach, you have 3 blood glucose
testing, which are FBS, after 1 hr of glucose load, and
after 2hrs of glucose load. Note: Nothing should be
spilled and the patient must take this within 5 minutes.
1 hour plasma glucose <140 mg/dL= no DM
1 hour plasma glucose >140 mg/dL= GDM possible,
proceed to the 3-h GTT (ADA)
The one step approach of OGTT is a screening test.
TABLE 6: ADA diagnostic criteria for 1-step approach
Fasting Blood Glucose ≥ 92 mg/dL (≥5.1 mmol/L)
(at least 8 hours
fasting)
1-hour OGTT 75 g ≥ 180 mg/dL (≥ 10 mmol/L)
2-hour OGTT 75 g ≥ 153 mg/dL (≥ 8.5 mmol/L)
Any one of the criteria is met = GDM
Oral glucose tolerance test two-step approach:
1. FBS is first tested.
2. 50 g (orange liquid) glucose load ( glucola) is given.
3. Test glucose after 1 hour. If value is 140 mg/dL (≥7.8
mmol/L), proceed to 3- hour OGTT using 100 g (clear
liquid) glucose load
4. Patient is subjected to overnight fasting after 3 days of
unrestricted diet of ≥150 g carbohydrate daily and
unlimited activity physical
3-hour OGTT reference values (2-step approach)
Fasting Blood Glucose ≥ 95 mg/dL (≥5.3 mmol/L)
(at least 8 hours
fasting)
1-hour OGTT 100 g ≥ 180 mg/dL (≥ 10 mmol/L)
2-hour OGTT 100 g ≥ 155 mg/dL (≥ 8.6 mmol/L)
3-hour OGTT 100 g ≥ 140 mg/dL (≥ 7.8 mmol/L)
Any two higher values of the 4 values = GDM
3-hour OGTT is the confirmatory test
Notes:
1. Patient is given a glucose preparation that should be
taken within 5 minutes.
2. Patient should rest throughout the test.
→ if the patient vomits, they are not qualified for the test
Requirements for OGTT
➔ Patient should be on a regular diet containing at least
150g of carbohydrates per day for 3 days
➔ Patients should not smoke, drink alcohol or coffee, or
exercise strenuously for 8 hours before or during the
test.
➔ Patients are allowed to fast overnight for 8-12 hours.
➔ Patient is given a glucose preparation that should be
taken within 5 minutes.
➔ Patient should rest throughout the test. If the patient
cannot tolerate the taste of the glucose load or heavy
malabsorptive syndrome or previous gastric or intestinal
surgery, it is best to use the IVGTT.
Intravenous glucose tolerance test (IVGGT) is indicated for
patients who have malabsorptive syndromes or previous gastric
or intestinal surgery. It is a poor method for estimating glucose
disposal because the disappearance of glucose in the blood is
slow
PROCEDURE OF IVGGT
1. Blood is drawn before infusion ( o minutes)
2. 20% glucose is administered IV within 3 minutes.
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3. The load is calculated at 0.5 g/kg body weight.
4. Blood is drawn after 1,3,5,10,20,30,40,60,120 minutes
5. Results are plotted in a graphing paper.
6. The glucose disappearance constants (K values) are
calculated from the plot of the logarithm of glucose
concentration against time.
7. There is DM if K value is <1.2
Glycosylated hemoglobin (HbA1c)
➔ Glycated hemoglobin method; common nowadays to
assess DM
➔ Glycosylated hemoglobin (HbA1c)Glycosylated
hemoglobin is also called total fasting hemoglobin.
➔ HbA1c is the most abundant adult hemoglobin. It is the
hemoglobin formed when glucose reacts with amino
group of hemoglobin.
➔ MECHANISM: The glucose molecule attaches non-
enzymatically to the hemoglobin molecule in a
ketoamine structure to form a ketoamine. The rate of
formation is directly proportional to the plasma glucose
concentrations.
➔ Therefore, HBA1c is a tool for long- term monitoring
for diabetes. The values provide information about the
average blood glucose level in the preceding (past) 2 to
3 months (since RBCs have 120-day life spans).
➔ Blood is collected WITHOUT fasting, but still, it will
show and provide the clinician a good data of the
glucose levels in the blood; moreover, compliance to the
medication and to the advice of the physician shall be
reflected in the result
➔ Studies show that lowering HbAIC to an average of less
than 7% reduces microvascular, retinopathic,
neuropathic complications of diabetes.
➔ NOTE: HB1Ac is expressed in %
TABLE 8: ADA diagnostic criteria for (HbA1c)
Normal Less than 5.7%
● hemoglobinopathy ( the hemoglobin will have
less time to become glycosylated and the
glycosylated hemoglobin level will be lower.)
● pregnancy (2nd and 3rd trimester)
● recent blood loss or transfusion
● hemolytic anemia
3. Thalassemia, chronic renal failure, dialysis patient,
splenectomy, elevated triglycerides or Hb F ( increased)
➔ ADA recommends that AIC is performed at least 2
times in a year to patients who are meeting treatment
goals and who have stable glycemic control. On the
other, patients who do not belong to the previous
statement must be tested every 3 months.
➔ The specimen requirement is an EDTA whole blood
sample. A hemolysate is prepared from this sample.
2 MAJOR METHODS FOR HbA1C
➔ Based on charged differences between glycosylated and
nonglycosylated hb
◆ Cation exchange chromatography
◆ Electrophoresis
◆ Isoelectric focusing
➔ Structural characteristics of glycogroups on hb
◆ Affinity chromatography
◆ Immunoassay
HbA1C Preferred Method
➔ affinity chromatography because it is NOT temperature
dependent and not affected by hemoglobin F, S or C
TABLE 9: THE METHODS OF MEASUREMENT ARE GROUPED
INTO 2 MAJOR CATEGORIES
A. Methods based on structural characteristics of
glycogroups on hemoglobin
Immunoassays Polyclonal or monoclonal
toward the glycated N-
terminal group of the ß-
chain of Hgb

Impaired 5.7% to 6.4%

Diabetes 6.5% or higher Affinity Separates based on Not temperature
chromatography( chemical structure using dependent
THERE ARE INTERFERING FACTORS TO HbA1c) preferred borate to bind glycosylated Not affected by
1. Failure to adequately mix the sample and the method) proteins other hgb
anticoagulant (EDTA tube is used for sample collection) like F,S, or C
2. Red blood cell life span. The shorter the RBCs, the ➔ This is the
lower the result. Conditions that decrease the lifespan preferred
of RBCs are: method
● anemia

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B. Methods based on charged differences (between
glycosylated & nonglycosylated hb)
Ion-exchange Positive charged resin
Chromatography
Electrophoresis Separation is based on
difference in charge
MIDTERM: S.Y. 2022-2023
definition for HbA1c and a reference method that
specifically measures the concentration of only one
Highly molecular species of glycated A1c, the glycated
temperature N-terminal residue of the β-chain of hemoglobin. The
Dependent. method uses either HPLC/electrospray mass
Affected by spectrometry or HPLC/capillary electrophoresis. This is
Hemoglobinnopat only used to standardized A1c and not used for clinical
hies measurement.
➔ A1c results are represented worldwide in IFCC units
High F values >7% (mmol HbA1c /mol hb) and derived NGSP units (%). This
interfere uses the master equation:
with analysis NGSP= [0.09148 x IFCC] + 2.152

Isoelectric Type of electrophoresis Pre-Hgb A1C Oral lactose tolerance

focusing using isoelectric point to interferes This test is used to measure plasma glucose levels after ingestion
separate of a challenge dose of lactose. It is used to screen for lactose
intolerance due to lactase deficiency (in the borders of small
High Form of ion-exchange Separates of all intestines). True congenital lactates deficiency is rare. Usually,
performance chromatography forms of lactose intolerance is acquired because lactase levels generally
liquid glycosylated hgb: decrease with age.
chromatography A1a, A1b,
A1c PROCEDURE FOR OLT
1. Collect blood after 8 hours fasting in a tube with
Estimated average glucose (eAG) in mg/dL can be derived from sodium fluoride and potassium oxalate.
AIC values using the formula: 2. Load the patient with 50 g lactose dissolved in 400 mL
HbA1c conversion to mg/dL water. For children 50 g/m2 of body surface area.
➔ (1.583 x HbA1c - 2.52) 18.05 3. Draw blood 30,60, and 120 minutes after giving the
➔ 28.7 x HbA1c - 46.7 loading dose.
4. Stool may be collected 5 hours after the loading dose.
TABLE 10: SAMPLES OF eAG (estimated average glucose) Note: It assesses intolerance to obtain information aside
eAG A1C (%) from that of the blood and from the stool.
5. Reference values: glucose levels rise over 20 mg/dL
97 mg.dL (5.4 mmol/L) 5
(>1.1 mmol/L) over fasting levels within 15 to 60
126 mg/dL (7 mmol/L) 6 minutes after ingestion of lactose.
154 mg/dL (8.6 7
mmol/L) Fructosamine
183 mg/dL (10.2 8 ➔ Fructosamine represents glucose complexed with other
mmol/L) proteins. Measurement is similar to glycosylated
212 mg/dL (11.8 9 hemoglobin, however, fructosamine is a measure of
mmol/L) short- term control of glucose.
240 mg/dL (13.4 10 ➔ It is an assay used to assess glycemic control over a
mmol/L) short period of time (6-8 weeks).
269 mg/dL (14.9 11 ➔ Fructosamine also results from non-enzymatic glycation
mmol/L) of protein with the formation of ketoamine-linked
298 mg/dL (16.5 12 glucose protein.
mmol/L) ➔ This is not very common
NOTE: md/dL to mmol/L = multiply by 0.055
➔ The International Federation of Clinical Chemistry and
Laboratory Medicine (IFCC) developed a common

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ADVANTAGES OF FRUCTOSAMINE
➔ It can be performed on automated machines.
➔ It is recommended in cases of hemoglobinopathies and
hemolytic anemias, when glycosylated hemoglobin
cannot be accurately measured.
➔ This is not affected by hemoglobinopathies, which is a
very common interference with HbA1c
DISADVANTAGES OF FRUCTOSAMINE
➔ Result is affected by changes in the level of serum
proteins. The assay should not be performed when
serum albumin level is 3 mg/dL or less.
➔ High uric acid and bilirubin levels and the presence of
heparin affect the test.
➔ There is a need to monitor fructosamine level monthly in
order to obtain information similar to that obtained from
performing HgbA1C every three months
1-deoxyglucose
➔ This is currently under investigation as a more sensitive
measure of glycemia than HbA1c or fructosamine.
➔ This better for DM
Tissue Oxidase test – Lactic acid and pyruvic acid
➔ Lactic acid, present in blood as lactate ion, is derived
primarily from muscle cells and erythrocytes.
➔ It is an intermediate product of carbohydrate metabolism
and is usually metabolized by the liver.
➔ Its concentration depends on the rate of production and
metabolism.
➔ Levels may significantly increase during exercise.
➔ Lactate and pyruvate form a reversible reaction that is
regulated by oxygen supply. When oxygen is deficient,
pyruvate converts to lactate. When oxygen is adequate,
lactate converts to pyruvate. When the hepatic system
fails to metabolize lactose sufficiently or when excess
pyruvate converts to lactate, lactic acidosis may result.
➔ Blood is collected using sodium heparin and potassium
oxalate.
➔ Reference values for blood lactate:
0.93-1.65 mEq/L ( 0.93-1.65 mmol/L);
pyruvate: 0.08-0.16 mEq/L (0.08 to 0.16 mmol/L)
The lactate-pyruvate ratio is less than 10:1.
Ketones
➔ Ketone bodies are produced in the liver through
incomplete fat metabolism (β- oxidation).
- Type 1 DM
➔ Ketones provide a ready energy from stored fatty acids
in times of low carbohydrate availability.
➔ The 3 ketone bodies are acetoacetic acid (20%);
ß-hydroxybutyric acid (78%); and acetone (2%).
➔ Ketones measurement is recommended for type 1 DM
patients during acute illness, stress, pregnancy, or
elevated glucose levels above 300 mg/dL
➔ Produced in CHO deprivation or decreased
carbohydrate use
➔ Ketonemia- accumulation of ketones in blood
➔ Ketonuria- accumulation of ketones in urine
- Lipids are being broken down in cases like Intermittent
fasting
Ketones can be identified and measured. The specimen
requirements are:
a. fresh serum or urine
b. covered container
c. immediate examination
Methods for ketone body do not identify ALL the ketones.
a. Gerhardt’s test (acetoacetic acid)
➔ uses ferric chloride to react with acetoacetic acid to
produce red color
➔ can react with interfering substances like salicylates
b. sodium nitroprusside (acetoacetic acid)
➔ reacts with acetoacetic acid in an alkaline pH producing
purple end color.
c. sodium niroprusside with glycerin detects acetone
(urine strips)
d. ß-hydroxybutyrate dehydrogenase detects
ß-hydroxybutyrate or acetoacetate depending on the
pH of the solution.
Note: at pH 7, the reaction proceeds to the right (decreasing
absorbance); at pH 8.5 to 9.5, the reaction proceeds to the left (
increasing absorbance)
Microalbuminuria
➔ micro=small, uria=presence in the urine,
microalbuminuria=small amounts of albumin in the
urine.
➔ Even the presence of minute albumin in the urine
signifies abnormalities.
➔ This is used to monitor diabetic inpatients (hospitalized)
➔ This is the presence of albumin in the urine of patients
with DM. Microalbumin is a useful test to assist in
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diagnosis at an early stage and before the development production. If insulin concentrations are greater than the
of proteinuria. Microalbumin concentrations are 20 to C-peptide concentration, the plasma insulin is from
300 mg/day. Proteinuria is typically greater than 0.5 exogenous administration
g/day.
➔ DM causes progressive changes to the kidneys that may TABLE 11: WHO DIAGNOSTIC CRITERIA FOR DM
result in renal nephropathy. TEST VALUES FOR VALUES INDICATING
➔ Can be detected through test for microalbuminuria IMPAIRED DM
(dipstick) CRITERION
➔ An early sign of nephropathy is an increase in urinary
Random blood > 200 mg/dL (11.1
albumin.
glucose or RBS mmol/L) + symptoms of
- Chemical parts include protein and sugar
(Random blood DM
- Non protein nitrogen = Creatinine
sugar)
Fasting blood 110-125 is impaired> 126 mg/dL (7.0
Microalbuminuria methods
glucose FBS mmol/L)
➔ Random spot collection for measurement of
2 hours 140-199 is impaired> 200 mg/dL (11.1
albumin-creatinine ratio(preferred)
(postprandial) glucose tolerance mmol/L) during an oral
➔ 24h collection
plasma glucose glucose tolerance test
➔ Timed 4h overnight collection
HbA1c >6.5% using a method
that is NGSP certified
THERE IS PERSISTENT ALBUMINURIA IF:
National glycohemoglobin standardization program
➔ In two out of three urine collections of 30 to 300
mg/24h, 20 to 200 μg/min.
LIPIDS AND LIPOPROTEINS
➔ Or an albumin/creatinine ratio of ≥300 mg/24h,
➔ Lipids and Lipoproteins are clinically important because of
>200μg/min
their association with CHD or coronary heart disease
➔ Or creatinine ≥300μg/mg
➔ Lipids are known to be insoluble in water(immiscible) and
can only be extracted using organic solvents; it is one of the
FACTORS AFFECTING MICROALBUMINURIA
dangerous substances in the body, but it also has an
➔ Exercise within 24h - positive urine protein important role in the body; associated with gluconeogenesis
➔ Infection - mostly caused by Streptococcus and the formation of ketone bodies
➔ Fever ➔ lipoproteins could carry lipids; they are lipids linked or
➔ Congestive heart failure combined with lipids
➔ Marked hyperglycemia ➔ Lipoproteins are carriers
➔ Marked hypertension ➔ Lipoproteins make up the body’s “petroleum industry”,
transporting fats for fuel needs while lipids perform these
Islet autoantibody, Insulin and C-peptide testing functions:
➔ Autoantibody testing is not currently recommended for a. serve as a primary source and storage of fuel
routine screening for diabetes diagnosis = because of the carbon-hydrogen bonds of
➔ Insulin is for hypoglycemic states and not diabetes. lipids that make them rich source of energy and
➔ Islet autoantibody testing is used to clarify the etiology capable of storing excess calories
of diabetes in patients with newly diagnosed diabetes. - Gluconeogenesis
➔ Insulin is not required to diagnose diabetes, however, in b. structural components of membranes due to
hypoglycemic states, it is important to know the its unique physical properties (phospholipid
concentration of insulin in relation to the plasma bilayer)
concentration. A decreased glucose with a c. cell surface components
non-suppressed insulin greater than 8 units at around
36 hours fasting suggests insulinoma.
➔ C-peptide assays have little diagnostic use, except in
instances in which inappropriate insulin administration
must be distinguished from endogenous insulin

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undergo metabolism in the liver which leads to the
production of ketone bodies.
➔ It should be noted that ketones are toxic to the body and
increased production leads to ketosis (an increased
ketones in the blood) or ketoacidosis (an extreme case of
ketosis where the body cannot regulate ketone
production resulting in lower blood pH).
➔ Most dietary fatty acids have straight chain compounds
with even numbers of carbon atoms. Examples are
palmitic (16 carbons); stearic, oleic, linoleic, & linolenic
(18 carbons)

→ There is an arrangement of fatty acids wherein the head is

hydrophilic and the tail is hydrophobic. There are also
glycolipids making up the cell membrane - making them a
substance that is one of the important components of this
membrane.
the carbohydrate structure detects the antigen present in the cell
(determines the antigenicity)
- Can be determined through blood typing
d. transport
e. Precursors Category of FA according to the number of double bonds in the
→ there are substances derived from lipids molecule:
a. saturated fatty acids– bonded with hydrogen atoms; no
➔ Lipids in the body are classified as double bonds present (ex. Palmitic acid)
◆ fatty acids, b. unsaturated fatty acids–contains single and double
◆ triglycerides, bonds. These are classified further as:
◆ phospholipids, 1. monounsaturated – one double bond (ex. Oleic
◆ sterols, acid)
◆ sphingolipids, & 2. polyunsaturated- two or more double bonds
◆ cholesterol (linoleic & linolenic acids)

BODY LIPIDS CLASSIFICATION

Fatty Acids
➔ Fatty acids are structural units of lipids. The kinds of fatty
acids could be classified according to the number of
carbon atom (length of c chain) or number of double
bonds present

Category of FA according to Number of Carbon Atoms:

➔ short-chain ( 4-6 carbon atoms)
➔ medium chain (8-12 carbon atoms)
➔ long-chain ( more than 12 carbon atoms) → Pointed by the arrow is an example of a monounsaturated
➔ These are the kinds of fatty acids that can provide twice fatty acid because it only contains a single double bond.
the energy as compared to carbohydrates. KINKS- double bond links that increase fluidity
➔ If carbohydrate is deficient like in the case of excessive
fasting/starvation or uncontrolled DM, fatty acids

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CATEGORY OF FATTY ACIDS (According to the number of
double bonds in the molecule)
Saturated No double bonds
Unsaturated One double bonds
● Monounsaturated -one double bonds
● Polysaturated - two or more double bonds
➔ The double bonds of unsaturated fatty acids are usually
arranged in cis form, which causes the fatty acids to bend.
These bends increase the space that the fatty acids require,
resulting in increased fluidity at room temperature or even
at 4˚C. Plants are good sources of these unsaturated fatty
acids. Meanwhile, saturated fatty acids from animals form
solid at room temperature.

→ Monounsaturated Fatty Acids: Omega 9 fatty acids —>

Oleic Acid
→ Polyunsaturated Fatty Acids:
Omega 3 fatty acids: Eicosapentaenoic Acid (EPA)
Alpha Linolenic Acid (ALA)
Docosahexaenoic Acid (DHA) ◆ Unsaturated is healthier due to the presence of double
bonds
Omega 6 fatty acids: Arachidonic Acid (AA) ● Double bond - kink of the structure
Linoleic Acid (LA) ● More double bond = more kink
Gamma Linoleic Acid (GLA) ● Clinical relevance of kink
Conjugated Linoleic Acid ( CLA) ○ Presence of double bonds would make the
lipids stay fluid
➔ Most fatty acids are synthesized in the body from
○ Lipid is healthier if
carbohydrate precursors but there are two fatty acids which
○ The more solid the fat is = greater risk of
are not synthesized in the body, thus, we need to take them
clot formation (this would activate the
through our diet because they are important for growth,
platelets)
maintenance, and proper functioning of many physiological
○ Saturated —> saturated fat
processes. We call them essential fatty acids. The essential
○ Animal fat = solidifies during cold temp
fatty acids are: linoleic acids and linolenic acid.
○ If saturated = easily solidifies
Essential Fatty Acids are:
➔ Essential fatty acid = essentially taken by the patient
through diet because it is not produced by the body

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◆ Must be essentially taken in the foods and
drinks because the essential fatty acid is not
produced by the body. ➔ Fatty acid C=C double bonds can also occur in trans
configuration, with both hydrogen atoms on opposite
sides of C=C double bonds.
- Because of the spatial orientation of their
double bonds, trans fatty acids do not bend and
have physical properties more similar to
saturated fatty acids.
- Trans fatty acids are not common in nature but
they can be present in our diet due to chemical
hydrogenation treatment used in food
processing to increase the viscosity of oils by
converting polyunsaturated plant oils into solid
margarine.
ESSENTIAL FATTY ACIDS
- The major dietary trans fatty acid is elaidic acid.
→cannot be synthesized in the body As epidemiological studies have shown,
➔ Linoleic acid and linolenic acid are essential fatty acids consumption of trans fatty acids increases the
under Omega 6 FA] risk of CHD.
➔ Should be essentially present in the diet (must be
taken from the diet as they are not produced by the
Triglycerides (TAG)
body)
➔ Also called triacylglycerol
➔ The molecule is composed of one glycerol and 3 fatty
Non-essential fatty acids, on the other hand:
acids, attached by ester bonds. Each fatty acid in the TAG
◆ PRODUCED by the body
molecule is a potential different structure, thus, one
◆ Does NOT mean less important
molecule of triglyceride can produce several types of
→BOTH are equally important
triglycerides. TAGs containing saturated fatty acids are
Essential fatty acids are:
packed closely and formed solids at room temperature
a. Linoleic acids while TAG containing unsaturated fatty acids may form oils
b. Linolenic acid at room temperature.
➔ TAG allows the body to store long carbon chains of
Forms of fatty acids in the blood: fatty acids needed during fasting states between
meals.
➔ Gluconeogenesis → noncarbohydrate sources of energy,
Free fatty acids This is otherwise called unesterified fatty
such as TAG
(FFA) acid (UFA) or non-esterified fatty acid
➔ The high energy TAG molecules are transported in the
(NEFA).
plasma in the form of chylomicrons and VLDL.
Component of TAG
It is the most metabolically active plasma
➔ When metabolized, their fatty acids are released to the
lipid and is bound to albumin
cells and converted to energy while glycerol is
→Albumin: a protein that would carry
recycled into additional TAG.
these fatty acids
➔ The breakdown of TAG is done by enzyme lipoprotein
Esterified fatty It is covalently linked with other lipase (LPL), and hormones epinephrine and cortisol
acid molecules such as TAG and cholesteryl LPL is also known as the Clearing factor; facilitates the
esters (cholesterol esters). This is the breakdown of TAG
majority of plasma fatty acids →RECALL, Epinephrine (produced by the adrenal medulla): fight
—-> esterified form: 3 fatty acids linked or flight response, Cortisol (produced by the adrenal cortex):
together (component of TAG) stress hormone

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● Epinephrine and cortisol will break down fat when
carbohydrate source are scarce
● The supply of free fatty acids to be used for TCA
cycle(Krebs cycle / Polycycle) depends on LPL interaction
with chylomicrons and VLDL
● TAG may be classified as
○ exogenous (diet)
○ endogenous (liver & other tissues)
CLASSIFICATION OF TAG
Exogenous: →taken from the food being eaten
Endogenous: →TAG produced by the liver and other tissues of
the body
● One of results why there is a need for fasting (12
hours)
○ Due to the accumulation of TAG in the body
because of the food we eat
● Inaccuracy in the number of fasting hours required for
triglycerides
→result: FALSELY ELEVATED TAG (in the blood)
→does not mean treatment is needed
→elevation of TAG only comes from the food eaten
➔ High-energy molecules produced by the body or from
the food we intake
◆ Could either be:
◆ Exogenous TAG
● From the diet/food we eat
● Rice → source of TAG
● Carried by the chylomicrons; the
moment exogenous TAG enters the
body, they are carried
chylomicrons
○ Rich in TAG → from the
foods and drinks taken
◆ Endogenous TAG
● Produced within the body
● note that even if we do not eat rice
(which is one of the culprits of rising
TAG), we still have endogenous
source
● Carried by the Very low density
lipoprotein
○ Serum/plasma = turbidity/creamy appearance
○ Especially if the food taken is rich in TAG
○ Cloudiness is due to chylomicrons that contain
TAG
○ This would explain why subjected to TAG testing
must fast for 12 hours, because if blood is taken
without fasting and the test is TAG you will be
measuring the recent intake of TAG (from the
food) not the real TAG in the body
◆ Fasting = TAG is clear up
→Recall in the carbohydrates lesson, Gluconeogenesis:
non-carbohydrates sources of energy which one is
Triglycerides
➔ Excess carbohydrates are stored as TAGs. Insulin
increases the synthesis of TAGs from carbohydrates.
Excess fatty acids in the blood are re-synthesized into
TAGs and stored in adipose tissues.
➔ Decreased TAGs are seen in hyperthyroidism.
➔ Increased TAGs is observed in DM, pancreatitis,
alcoholism, glycogen storage diseases, hypothyroidism,
nephrosis, gout, and use of oral contraceptives.
➔ TAG in the body could be exogenous (dietary) or
endogenous (synthesized in liver and other tissues).
Phospholipids
➔ is an amphipathic property (has polar hydrophilic
[water-loving] head groups and nonpolar hydrophobic
[water-hating] fatty acid side chains).This property allows
the molecule to act as detergent, a characteristic suited to
its function as lipid membrane.
➔ (does not allow any substances to pass through; selective
permeability)
➔ Has amphipathic property that allows the molecule to act
as detergent, a characteristic suited to its function as lipid
by membrane
◆ Amphipathic means that the molecule has polar
hydrophilic or water-loving head and the
hydrophobic or water-hating fatty acid chains
or tail
➔ Forms a coating that surrounds cholesterol & TAG and
"glues" them to lipoprotein core
Other important functions of phospholipids:
● Most phospholipids are formed by the union of 2 fatty
acids, a phosphorylated glycerol (lipid attached to a
phosphate), and a nitrogenous base (categorization of
one lipid to another).
● The nitrogenous base differentiates one phospholipid
from the other.
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❖ Niemann-Pick disease → a deficiency in
sphingomyelinase and accumulation of sphingomyelin;
❖ Gaucher’s disease→ a deficiency in ß-D – glucosidase
and accumulation of glucocerebroside.

Sterols
➔ are made up of ringed structures connected to a long
aliphatic chain with at least one hydroxyl group. Plant
sterols resemble like cholesterol; they can be used to reduce
absorption of cholesterol.

Cholesterol
➔ is the major unsaturated steroid alcohol in humans. It
contains a perhydrocyclopentanthroline ring and a side
chain of 8 carbon atoms.
PHOSPHOLIPIDS IMPORTANT FUNCTIONS ➔ Can be termed as cholesteryl esters
● major component of surfactant that allows the alveoli ➔ Most serum cholesterol is in the form of cholesterol
to distend during breathing esters (cholesteryl esters).
● for mitochondrial metabolism ➔ Cholesterol esters are transported by LDL and HDL
● blood coagulation
● lipid transport.
◆ Exogenous
◆ Endogenous
Sphingolipids ◆ If moved from the liver to peripheral tissues → carried
➔ is a lipid membrane component of RBCs, brain and nerve by LDL
cells. It is NOT derived from glycerol but from amino ● More prone to cardiovascular disease
alcohol, called sphingosine. (coronary heart disease)
◆ Difference from phospholipid = does not have ● LDL = bad cholesterol
glycerol ◆ If the Cholesterol is moved from circulation/peripheral
➔ It is important in cell recognition and blood typing: nerve tissues back to the liver, it is carried by HDL
transmission. ● Explain why HDL is termed as good
➔ A glucosylceramide is an example of sphingosine. The cholesterol
types of ceramide derivatives in humans are called ● It gets the unused cholesterol and keep them
glycolipids or sugar containing lipids: to the liver
GLYCOLIPIDS OR SUGAR CONTAINING LIPIDS ● Elevation of enzyme = storing of cholesterol
sphingomyelin in the liver
contains a phosphocholine group
◆ The body normal gets cholesterol from the liver to the
The 2 simplest glycolipids are body
galactosylceramide contains galactose ● Cells in the body needs cholesterol
glucosylceramide contains glucose (glycolipid or glucose ○ (refer back to the functions of
containing lipid) cholesterol)
● LPL = lipoprotein lipase
Sphingolipid Diseases ○ Clearing agent of TAG
● Lipid storage diseases are inherited deficiencies of
sphingolipid catabolic enzyme (sphingomyelinase). FUNCTIONS
These diseases are characterized by the accumulation of a. structural component
sphingolipids in the brain that may lead to death in b. manufacture of hormones and repair of tissues
children. These are :
c. synthesize of bile acids and vitamin D
If the enzyme is deficient —> accumulation of substrate

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d. precursor of 5 major classes of steroid hormones: ● During Insulin deficiency, the Heart and brain
● Progestins still has ENOUGH supply of insulin and
● Glucocorticoids glucose
● Mineralocorticoids ● However, if there is unavailability of GLUCOSE
● Androgens —> present in both male and female, but (fasting, hypoglycemia) then Ketone bodies
elevated in male would help supply the said organs with energy
● estrogens. ➔ Produced by the liver mitochondria from the acetyl coA
when excessive amounts of fatty acids is limiting.
NOTE: CHOLESTEROL COULD NEVER BE A SOURCE FOR ATP ◆ Note that ketone bodies are produced, but
NOT UTILIZED in the liver
CHOLESTEROL COULD BE (found almost exclusively in ➔ Products if fats are broken down by the body
animals)
a. exogenous – 100 to 700 mg per day Lipoproteins
(dietary) ➔ Transport vehicles for lipids
b. endogenous 500 to 1000 mg/day ➔ contain TAGs, cholesterol, phospholipids and
(hepatic and ➔ 600 to 1000 mg Biliary apolipoproteins. Lipoproteins are classified by either
other tissues) cholesterol is also secreted into their density ( the more lipids, the lower is the density)
the intestine with 50% of this as well as the apolipoproteins attached on their surface.
reabsorbed into the blood ➔ The major classes according to the density are:
chylomicrons, VLDL, intermediate-density LDL, and
TWO TYPES OF CHOLESTEROL HDL.
a. free cholesterol – is around 30%.
(metabolically active) →It is a non-esterified form, and has MAJOR TYPES OF LIPOPROTEINS
an amphipathic property. Chylomicrons ➔ are the largest and least dense
→It forms the mobile form. lipoproteins.
b. esterified cholesterol → most abundant ➔ It can float
– is around 70% of cholesterol ◆ Component the floats in
→ is located in stationary pools the plasma/serum
located in the skin, adipose tissue, ➔ They are the major carriers of
and muscle cells. exogenous triglycerides to
→It is covalently linked with one muscle cells and adipose tissues.
fatty. →TAG derived from the food that is eaten
→The molecule is not anymore (dietary) are carried by chylomicrons
amphipathic but already ➔ Chylomicrons originate in the
hydrophobic. intestinal tract and travel to the
blood and lymph to various
tissues. They are NOT normally
Ketone Bodies
found in 12-hour fasting blood
➔ THREE KETONE BODIES
sample
◆ Acetoacetate
➔ They are synthesized in the small
◆ B-OH-butyrate (major ketone body),
intestine.
◆ acetone
➔ Degraded in the liver
➔ derived from Fats
➔ They are not normally found in
→in DM, metabolic acidosis causes acetone breath due to the
12-14 hour fasting blood sample
ketone bodies
→reason: needed to fast for the Lipid
➔ The ketone bodies supply energy to the heart, skeletal
profile
muscles, and the brain.
→necessitates for 12-14 hour fasting for
→HEART & BRAIN are not dependent on the amount of the
TAG (lipid profile)
INSULIN in the body

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➔ Very big and less dense in terms
of size
COMPONENTS OF CHYLOMICRONS
❖ TAG :90-95% (major composition)
❖ Phospholipids: 2-6%
❖ Cholesteryl ester: 2-4%
❖ Free cholesterol: 1%
❖ Apolipoprotein: 1-2%
➢ Apo C and apo B-48 are the most common
abundant apolipoproteins
➢ Apo E, apos A-1,II, and IV are found in small
amounts
➢ They would change from one lipoprotein to
another
Very Low-Density ➔ Smaller than chylomicrons
Lipoproteins (VLDL) ➔ Rich in TAG and are the major
carriers of endogenous TAG to
muscle and adipose tissues.
➔ They are synthesized in the liver
and smaller and denser than
chylomicrons.
◆ They are degraded in to
LDL in the circulation
→Contain equal amounts of phospholipids
& cholesterol
→Contain apoB-100, apoC, apoE
➔ A large carbohydrate intake
enhances= TAG liver synthesis
(elevated TAG production)=
increases VLDL production.
◆ Increase rice intake =
increase TAG =
increased VLDL and
chylomicrons
➔ An increased VLDL and
chylomicrons produce a turbid
serum.
➔ Chylous, milky serum is
attributed to the elevated VLDL
and chylomicrons
Intermediate-density ➔ are formed when VLDL loses its
lipoprotein (IDL) TAG molecule.
Low-Density ➔ are formed when TAGs are
Lipoprotein (LDL) removed from the VLDLs.
➔ They are synthesized in the liver
and are the most cholesterol rich
of the lipoproteins.
➔ They are taken into cells via a
special cell surface, the
apoprotein B receptor which are
smaller than the TAG rich
lipoproteins
➔ LDL is smaller compared to
chylomicrons and VLDL
◆ Chylomicrons carry the
exogenous triglycerides
◆ VLDL carry the endogenous
triglycerides
➔ Carries cholesterol to the liver
for bile formation, to the tissues
for use in cell membranes, to
endocrine organs for steroid
hormone production, and to
cholesterol sites.
➔ Contain equal amounts of
phospholipid and protein
◆ Contain very little TAG
➔ LDLs are responsible for the
transport of cholesterol from
the liver to the peripheral
tissues,
◆ The reason why LDL is
called as the bad
cholesterol is because it
allows cholesterol from
the liver to go out to
the peripheral tissues
➔ They are the most atherogenic
lipoproteins and high levels are
attributed as a major CHD
(Coronary Heart Disease) risk
factor.
High-Density ➔ are the smallest and densest
Lipoproteins (HDL) lipoproteins; have higher
proteins and phospholipid
content of all lipoproteins.
➔ They contain mostly proteins,
some cholesterol, & little TAG
➔ They are synthesized by the
intestines and the liver.
➔ They can also be formed through
the catabolism of chylomicron
and VLDL particles.
➔ They take excess cholesterol
from the tissues and return it to
the liver.
➔ They keep the cholesterol in the
liver and will not allow them to
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stay in the blood, this is the
Lipoprotein (a) ➔ are LDL like lipoproteins particles
reason why HDL are called as
[Lp(a)] with one additional molecule of apo
the good cholesterol
(a) linked to apo B-100 by a disulfide
➔ Contain mostly apoA-1 and
bond.
apoA-II
➔ The presence of elevated serum levels
➔ And a little of apoC and ApoE
is an independent risk factor for the
➔ Major subclasses: HDL2 and
development of pre-mature CHD,
HDL3
myocardial infarction, and
cerebrovascular disease.
Comparison by Size of Lipoproteins

LpX Lipoprotein

Lipoprotein X ➔ an abnormal lipoprotein present in

patients with biliary cirrhosis or
cholestasis and in patients with
mutations in lecithin-cholesterol
acyltransferase (LCAT)
➔ Contains mostly of phospholipids,
unesterified cholesterol, & little
esterified cholesterol
➔ Has apoC & albumin
➔ differs from other lipoproteins because
it lacks apo B-100
➔ mainly removed by RES of liver and
spleen
➔ ApoC and Albumin
➔ typically included in the
LDL-cholesterol value as calculated by
Friedewald equation.

Apolipoproteins

Apolipoproteins ➔ Protein moieties associated with

(apo) plasma lipoproteins
Lipoprotein (a) [Lp(a)]
◆ are protein portions of
lipoprotein molecules.
➔ They are amphipathic molecules
that help to keep the lipids in
solution during circulation through
the bloodstream.
➔ It also regulates plasma lipid
metabolism by activating and
inhibiting enzymes that are involved
in the process.
DIFFERENT TYPES OF APO
a. Apo A ➔ this the major protein component
of HDL

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Apo A-1: major activator of the LCAT
➔ 75% of ApoA: synthesized in the
liver & intestine
➔ Activator of lecithin cholesterol
acyltransferase which esterifies
plasma cholesterol
Apo A-ll: activates hepatic lipase
➔ Unknown role
Apo A-lV: cofactor for LCAT
b. Apo B ➔ this is the major protein
component of LDL; insoluble in
water
Apo B-100 binds to cell receptor to target LDL to LDL
receptors or Apo B/E receptors
➔ Major component; synthesized in
liver
Apo B-48 structural role in chylomicrons
➔ Intestinal origin
c. Apo C ➔ this is the major protein component
of VLDL
Apo C-1 may activate LCAT
➔ Major constituent of chylomicrons,
VLDL, & HDL Physiology of Lipids
Apo C-ll potent activator of LPL
➔ Major component of VLDL; less than
3 Phases
2% in HDL
1. Digestive Phase
Apo C-lll regulates rate of clearance of TAG-rich
➔ Begins with chewing and swallowing
lipoprotein remnants
➔ Cholesterol becomes surrounded by bile to form a
➔ Major component of VLDL & HDL
micelle package that is absorbed by the small intestine
➔ Inhibits lipolysis of TAGrich
➔ TAG are digested by enzymes, bile salts, and acid in the
lipoproteins
gut to form monoglycerides and diglycerides
d. Apo D ➔ this is also called Apo A-lll or 2. Absorptive Phase
“thin-line apoprotein”. ➔ Occurs in the small intestine as TAG & cholesterol in the
➔ A minor constituent of HDL protein micelles are absorbed and broken down into fatty acids
and a Cofactor for LCAT 3. Transport Phase
e. Apo E ➔ this is rich in amino acid arginine. It ➔ Occurs as long as fatty acids reassemble into
recognizes cell receptors to target chylomicrons (water soluble macromolecules) and enter
chylomicrons and VLDL remnants to the lymphatic system
the hepatic receptors. ◆ This refers to the transport of fatty acids (other
➔ Arginine-rich apolipoprotein found or longer fatty acids)
in VLDL, IDL, remnant proteins, ➔ Short fatty acids enter the blood bound to
chylomicrons, & HDL albumin-heading to all tissues, including adipose tissue
➔ Have crucial role in the (where they can be stored)
pathogenesis of Alzheimer’s
disease Lipid Digestion
➔ An average person ingests, absorbs, resynthesis, and
transports 60 to 130 grams of fat daily in the body. Most
of this is in the form of triglyceride.

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➔ Fats are insoluble in an aqueous environment; hence,
emulsification must first take place. Emulsification
happens in the duodenum. Bile is released by the
gallbladder under the influence of cholecystokinin
hormone and is considered as the emulsifying agent. It
contains conjugated bile acids, phosphatidylcholine, and
cholesterol. Emulsification converts dietary lipids into S.
In micelles, the amphipathic lipids orient themselves
with the hydrophobic regions on the inside and the polar
groups exposed to the aqueous system.
➔ TAG and cholesterol esters are not amphipathic, hence,
can not form micelles. TAG hydrolysis in the small
intestine is under the enzymatic effect of pancreatic
lipase. Lipase activity is halted once the enzyme comes
in contact with micelles, hence, colipase, helps to
maintain its stability and anchor it onto micelles.

➔ On the other hand, phospholipids are hydrolyzed by

pancreatic phospholipase A.

NOTE!!
➔ Triglyceride and cholesterol esters are not amphipathic,
and hence, cannot form micelles
➔ Meanwhile, dietary cholesterol is a mixture of
➔ Lipase activity is halted or stopped once the enzyme
cholesterol and cholesterol esters. It is cholesterol esters
comes into contact with micelles, and hence, there is a
that is the most non-polar lipid and the most abundant
need for another enzyme, which is co-lipase. Co-lipase
form that accumulate in the arterial wall in
helps maintain stability and anchoring of enzymes into
atherosclerotic lesions.
the micelles
● Lipid absorption happens when the micellar solutions
➔ Dietary cholesterol is a mixture of cholesterol and
come in contact with microvilli of the mucosal cells.
cholesterol esters -the cholesterol esters that is the
● TAG and cholesterol esters are released into the lymph
most nonpolar lipid and most abundant form that
in the form of chylomicrons. Chylomicrons make blood
accumulate in the arterial wall in atherosclerotic lesions
turbid. TAG from the chylomicrons are cleared from the
circulation by lipoprotein lipase (lipemia clearing factor).
➔ Lipid absorption happens when the micellar solutions
● The fatty acid released from TAG then enters the
come in contact with the microvilli of the mucosal cells
extrahepatic tissues. The chylomicron left is degraded in
(intestine)
the liver. Hypertriglyeridemia normally occurs at its peak
➔ The fatty acid released from TAG then enters the
within 3-4 hours after a meal and completely cleared
extrahepatic tissues
within 6-8 hours in normal persons.
➔ The chylomicron left is degraded in the liver
➔ Hypertriglyceridemia (increase of TAG) would be
experienced at its peak within 3-4 hours after a meal,
and completely cleared within 6-8 hours in normal
individuals
◆ 12 hours of fasting must be completed for lipid
profile as there are persons who would have
their chylomicrons, TAGs cleared 8 hours after

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meal, while, there are those which would take
even 10 hours
➔ Reesterification involves re-synthesis of the fatty acids
and 2 –MAG to form back to TAG. For cholesterol, it is
formed from 3 molecules of acetyl-CoA which have
undergone a condensation process.
NORMAL LIPOPROTEIN METABOLISM
4 pathways for lipid and lipoprotein metabolism
A exogenous lipoprotein metabolic pathway,
B. endogenous lipoprotein metabolic pathway
C. Low-density lipoprotein receptor pathway
C. reverse cholesterol transport (HDL cholesterol pathway)

MEMORIZE !
HUMAN LIPASES A. EXOGENOUS LIPOPROTEIN METABOLIC PATHWAY
Types Source Action Properties
➔ This pathway transports dietary lipids, mainly TAG,
Pancreatic Pancreatic juice Digestion of Hydrolyzes TAG through the formation of chylomicrons in the small
lipase dietary TAG in mixed intestine.
micelles; ➔ The CMs are encapsulated in secretory vesicles in the
activity is Golgi apparatus.
enhanced by ➔ The cell uses exocytosis to transport packaged
colipase chylomicrons to the extracellular space The packaged
Hormone-sensi Adipocytes Fat mobilization Activated by CMs acquire additional apo C and apo E from circulating
tive lipase phosphorylatio HDL to complete the CM molecules.
n through the ➔ With the apo C-II attached to the surface of the CM,
action of Lipoprotein lipase (LPL) on the surface of the endothelial
cAMP-depende cells is activated.
nt protein ➔ TAG inside the CM are quickly hydrolyzed into fatty
kinase acids.
Acid lipase Lysosomes Intracellular pH optimum of ➔ The fatty acids attached to albumin for uptake into
catabolism of about 5.0 muscle and adipose cells. At the same time, circulating
lipoproteins HDL molecules absorb phospholipids and apo A from
Lipoprotein Capillaries Utilization of Released into the CM remnants.
lipase (LPL) TAG in plasma by ➔ The apo B-48 and apo E on the CM remnant act as liver
lipoproteins heparin, cell receptors, allowing the remnant to be taken by the
inhibited by liver cells.
protamine; also ➔ The components of the remnant are broken down into
called lipemia cholesterol, which is made into bile acids, newly
clearing factor synthesized lipoproteins, or cholesterol esters.
Hepatic lipase Liver Lipoprotein Released into
catabolism plasma by
heparin,
resistant to
protamine

Bile
➔ Produced under the influence of cholecystokinin
hormone and is considered as the emulsifying agent.
Bile contains conjugated bile acids: phosphatidylcholine
cholesterol and it functions for emulsification to convert
dietary lipids into mixed micelles.

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Newly synthesized chylomicrons in the intestines are initially interaction with apo B or apo E.
secreted in the lacteals. Lacteals are the lymphatic vessels in the
small intestine. The chylomicrons would then be passed in the
lymphatic ducts and eventually enter the circulation through the
thoracic ducts. After entering the circulation, the chylomicrons
interact with proteoglycans such as the heparin sulfate. The
proteoglycans along with specific protein the GP1HBP1 on
capillaries also promotes the binding of LDL which hydrolyzes the
TAG chylomicrons. The free fatty acid and glycerol generated are
taken up by the cells and used as energy. The excess fatty acids
in the adipose cells or adipocytes are reesterified into TAG for
long term energy storage. APO C2 found in the VLDL is important
in the activation of LPL or lipoprotein lipase. Hormone sensitive
lipase which is found in the adipocytes releases the free fatty
from the TAG in stored fats during fasting. During fasting Most of the TAG package into LDL in the liver comes from dietary
gluconeogenesis would occur. It is important that hormone sources. The VLDL once secreted into the circulation undergoes a
sensitive lipase would be stimulating the release of energy from lipolytic process similar to chylomicrons. VLDL loses core lipids
the stored adipose cells to be used as sources of energy. causing dissociation and transfer of apolipoproteins and
Epinephrine and Cortisol mobilize and hydrolyze the TAG from phospholipids to other lipoprotein particles such as the LDL
adipocytes while insulin, produced by the pancreas after a meal, primarily from the action of LPL(lipoprotein lipase), resulting in
prevents lipolysis by adipocytes and promotes fat storage and the conversion of VLDL to denser particle IDL or intermediate
glucose utilization. Fat storage is needed for whatever glucose we density lipoproteins, or VLDL or very low density lipoprotein
have to be utilized by the body. During lipolysis, breaking down of remnants. IDL persists for a short period and receives cholesterol
fats and chylomicrons, there is transfer of lipids and ester from HDL in exchange via the cholesteryl ester transport
apolipoproteins A1 and C2 into HDL and chylomicrons are protein. IDL is also taken up by the liver via APO E and LDL
converted a few hours after a meal into chylomicrons remnants receptors. The TAG in IDL are removed by the hepatic
particles. The chylomicron remnants are taken up rapidly by the triacylglycerol lipase and about half of the VLDL is completely
liver cells through the interaction of APO E with LDL receptor and converted to LDL and the remainder is taken up as VLDL
LDL related receptor which is found at the surface of the liver remnants.
cells. Once inside the liver, lysosomal enzymes would break down Low-density Lipoprotein Receptor Pathway
the remnant particles to release the free fatty acid, free
cholesterol and amino acids. Some cholesterol is being converted
to bile acids.

B. ENDOGENOUS LIPOPROTEIN METABOLIC PATHWAY

➔ This is responsible for transporting hepatic lipids by way

of VLDL and LDL to the peripheral tissues.
➔ VLDL particles, once secreted into the blood stream,
undergo a similar degradation process as CMs. Through
the action of LPL and apo C-ll, VLDL lose their core
lipids, mainly TAG, and also lose some of the
apolipoproteins and phospholipids from the surface.
During this process, VLDL become VLDL remnants, The LDL particles are responsible for the delivery of exogenous
which can then be converted to intermediate-density cholesterol to peripheral cells due to efficient uptake of LDL by
lipoproteins (IDL) and then further processed to LDL. the LDL receptors. The LDL receptors must be functional in order
➔ However, only about 50% of VLDL are converted to LDL; for the process to be effective. Once bound to LDL receptors, they
the other 50% are taken up by either the remnant are endocytosed by cells and transported to the lysosomes where
receptor or the LDL receptor of the liver through they are degraded. TAG in LDL are converted to acid lipase into
free fatty acids and glycerol and further metabolized by the cell

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for energy or are re esterified and stored for later use. The
regulation of cellular cholesterol biosynthesis is impart
coordinated by the availability of cholesterol delivered by the LDL
receptor. Abnormalities in LDL receptor function result in
elevation of LDL into the circulation that would lead to the
condition hypercholesterolemia and premature atherosclerosis.
Hypercholesterolemia increases cholesterol in the blood.
● Cell receptors recognize and bind to the apo B-100 of
LDL molecules.
● Then the bound LDL is taken into a vesicle that fuses
with the LDL to become an endosome. At this point, the
LDL receptors separate from the LDL and travel back to
the cell surface.
● The LDL migrates to a lysosome, where the apo B-100
is broken down into peptides, amino acids, and
cholesterol.
● Free cholesterol derived from degraded LDL can be used
in cell membranes for hormone synthesis or can be
re-esterified for storage.
● Cells regulate the amount of free cholesterol by
decreasing the amount of endogenous cholesterol
produced, increasing the production of cholesterol
esters, and inhibiting LDL synthesis when cholesterol
levels are high.
● Excess cholesterol esters are taken up by macrophages.
When macrophages become engorged with cholesterol
esters, they are called foam cells. Foam cells are the
earliest indication of atherogenic lesions.
● Abnormalities in the LDL receptor mechanism can lead
into elevations of LDL in the circulation and thus lead
into hypercholesterolemia and premature
atherosclerosis.
● The interaction between apolipoproteins and the
receptors on the surface of the cells is important for
2 main reasons
a. delivery of lipoprotein into the cell
b. efficient removal of atherogenic lipoproteins from the blood
and peripheral tissues.
3 MAJOR RECEPTORS
LDL receptor recognize both apo E and apo B-100 and mediate
cellular binding, uptake, and degradation of LDL,
VLDL, IDL
remnant recognize apo E and are the major receptors for
receptor the clearance of chylomicron remnants and
ß-VLDL from blood circulation. They can also bind
apo E-containing HDL
Scavenger : are found on the surface of macrophages and
receptors: muscle cells. They mediate the removal of
modified LDL, including oxidized LDL and ßVLDL
from blood circulation.
Individuals can be classified into 2 distinct phenotypes based
on LDL: type A and B
Type A
➔ Have large buoyant LDL particles
Type B
➔ Have smaller LDL
➔ Small dense LDL particles have been found to be an
independent predictor of coronary heart disease and is
also associated with increased risk of metabolic
syndrome. Small dense LDL particles have longer
half-life in the circulation than the normal LDL and are
more likely to be modified and taken up by the
scavenger receptors on macrophage, which will develop
into foam cells. The formation of the foam cells is one
critical step in the development of atheroma. Atheroma
is the degeneration of the walls of the arteries caused
by accumulated fatty deposits and scar tissue leading to
the restriction of the circulation and the risk of
thrombosis.
c. REVERSE CHOLESTEROL PATHWAY
➔ HDL is secreted from the liver as crescent-shaped
molecules containing mostly phospholipids and apo A-1.
➔ HDL molecules gain their spherical shape as they absorb
more phospholipids, cholesterol esters, and certain
apolipoproteins in the extracellular space.
➔ Hepatic cells can take up cholesterol in HDL in three
ways:
◆ HDL receptors can attach to HDL cholesterol
esters and transfer the cholesterol esters to the
liver
◆ HDL remnants can provide the cholesterol
esters to lipoproteins containing apo B-100
◆ apo E present on HDL remnants can be
recognized by liver cells
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HDL Cholesterol Pathway
HDL is good cholesterol. It removes excess cholesterol in cells in
multiple pathways. In the accused Diffusion pathway, HDL acts as
a sink for the small amount of cholesterol that can be diffused
away from cells. Cholesterol is amphipathic and is soluble in
micromolar amounts and can spontaneously dissociate from the
surface of cell membranes and enter the extracellular fluid. Some
free cholesterol binds to nascent HDL in the extracellular space.
Once it is bounded, it becomes trapped in lipoproteins after it is
converted to cholesterol ester cholesteryl ester by elka which
resides on nascent HDL and is activated by its co factor APO A1
and the nascent HDL is first converted to HDL 3. The formation of
cholesterol ester via elka increases the capacity of the surface of
HDL 3 particles to absorb more free cholesterol from cell
membranes. This is the reason why it is known as good
cholesterol. The absorption of free cholesterol from the cell
membrane along with APO C1,2,3, APO E, phospholipids from
VLDL and chylomicrons are converted to HDL2. HDL2 can
directly deliver cholesterol to the liver by the scavenger type B1
or SR-B1. This would deliver cholesterol to the liver. Cholesterol
that reaches the liver is then directly excreted into the bile or first
converted to bile acid before excretion. Another pathway where
HDL removes cholesterol from the cell (2nd pathway). The 2nd
pathway involves the ABCA transporter which transfers a lipid,
enabling APO A1 that has dissociated from HDL to bind to the
cell membrane. APO A1 has a detergent-like mechanism to
remove excess cholesterol from the plasma membrane to form a
discoidal-shaped HDL particle. The newly formed HDL is
competent to accept additional cholesterol by an accused
diffusion pathway and converted into spherical HDL by LCAT.
Recent studies show that ABCG1,ABCG5, ABCG8 transporters
have been found to facilitate the efflux of cholesterol to lipid-rich
spherical HDL via a mechanism that appears to be different from
the ABCA1 transporter.
ENZYMES IN LIPOPROTEIN METABOLISM
Lipoprotein It is found on the surface of capillary endothelial
Lipase (LPL) cells of adipose tissue and of skeletal and heart
muscles. It hydrolyzes TAG in chylomicrons and
VLDL
Lipolytic Barely detectable in fasting sample
enzymes
Hepatic TAG Has limited capacity to hydrolyze TAG in intact
lipase (HTGL) chylomicrons and VLDL
Lecithin-chole Catalyzes the esterification of cholesterol by
sterol promoting transfer of fatty acids from lecithin to
acyltransferas cholesterol. This results in the formation of
e (LCAT) lysolecithin and cholesterol ester
EFFECT OF HORMONES
Insulin inactivates lipase, suppresses release of free fatty,
in the liver; it regulates VLDL synthesis.
Growth increases production of VLDL, stimulates
hormone synthesis of LDL receptors, and enhances the
clearance of lipoproteins by the liver.
Sex hormones estrogen raises VLDL (exemplified by estrogen)
Thyroid stimulates LPL and hepatic lipase synthesis,
hormones increases LDL receptor production
GUIDELINES NECESSARY FOR BLOOD LIPID MEASUREMENT
Fasting: the ideal fasting time for lipid measurement is 12-14
hours
Position: Change in patient position results in a ~ 10% decrease
in total cholesterol, LDL-cholesterol, HDL-cholesterol, apo A-1
and Apo B. A 50% decrease in TAG is also observed
- If the patient is already sleeping, do not let him/her
stand and go back to the bed. Find a way to properly
obtain the blood with the patient’s current position
Tourniquet
- Prolonged application would result to lipid
concentration
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Sample: serum; plasma
- Serum is preferred, most especially for cholesterol, TAG,
and HDL-C measurements using chemical methods
- Plasma is preferred when measuring lipoprotein by
ultracentrifugation or electrophoresis
Anticoagulant. EDTA was the traditional choice for lipoprotein
separation because it was thought to enhance stability by
chelating metal ions
- However, studies have shown that microclots formed in
EDTA unseparated sample from cells during storage can
clog sample probes, which could damage the machine
- EDTA also osmotically draws water from RBCs, diluting
the plasma constituents
NCEP recommended cholesterol measurements made on EDTA
plasma require correction by factor 1.03
Storing: -200C for shorter storage and -700C or lower for
long-term storage
Abnormal Lipoprotein Metabolism
● Genetic defects in lipoprotein metabolism usually
involve defects in surface apoproteins of the
lipoprotein molecules, cell surface receptors of the
liver and peripheral cells, or enzymes that regulate
synthesis and catabolism. The table below shows
Fredrickson classification of lipid disorders.
● Both type of amount of proteins are abnormal
Hyperlipoproteinemia
➔ Hyperchylomicronemia is characterized by excessive
chylomicrons. Causes are:
◆ Familial lipoprotein lipase deficiency
◆ Familial apo C-11 deficiency
➔ Familial hypercholesterolemia is characterized by
excessive cholesterol due to high LDL. High LDL results
from low LDL receptors. Xanthomata is common
◆ Xanthomata means fatty deposits in tissues
under the skin
Familial Dysbetalipoproteinemia
➔ Familial Dysbetalipoproteinemia (remnant
lipoproteinemia - high abnormal lipoproteins (B-VLDL),
IDL, chylomicron remnants, TAG, cholesterol.
➔ Biochemical abnormality in apoE.
➔ Eruptive Xanthomas are common.
Familial Hypertriglyceridemia
➔ Increased VLDL & TAG (type IV) due to overproduction
of VLDL or decreased catabolism of VLDL. It is
associated with obesity.
● Usually associated when gaining weight or
getting fat

Type V Hypertriglyceridemia
➔ Characterized by elevated levels of VLDL &
chylomicrons

Familial combined hyperlipedemia

➔ Elevation of VLDL or LDL or both (Type IIb). Apo B - 100
is usually elevated

Hyper a-lipoproteinemia
➔ Increased HDL

Hypolipoproteinemia
➔ Caused by genetic defect leading to absent or decreased
LDL and HDL
➔ Type I: Familial Hyperchylomicronemia
➔ Type IIa: Familial Hypercholesterolemia
➔ Type IIb: Familial Combined Hypercholesterolemia
➔ Type III: Familial Dysbetalipoproteinemia
➔ Type IV: Familial Hyperlemia
➔ Type V: Endogenous Hyperglyceredemia

➔ Dyslipoproteinemia is characterized by quantitative and

qualitative abnormalities of plasma proteins

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● Steatorrhea = presence of fat in stool

Abetalipoproteinemia
➔ Defect in apo B synthesis. Chylomicrons, VLDL, LDL are
absent.

Familial B-lipoprotein deficiency

➔ Partial Apo B deficiency. Low LDL
Familial hypo-a-lipoproteinemia (Tangier’s disease)
➔ HDL deficiency. There is accelerated apo A-1 catabolism
● Children and adults have developed earlier
hypercholesterolemia
Lipid Disorders
➔ Usually lead to abnormal lipid deposits on walls of
vasculature (atherosclerosis) and skin (xanthomas)
➔ Hyperlipidemia obstruction leads to lack of bile, so
cholesterol cannot be adequately absorbed by the small
intestine

● TAKE NOTE: Laboratories can also have differences in

reference range but there would be a common number
● SI conversion factor for cholesterol = 0.026
● SI conversion factor for TAG = 0.0113

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CLASSIFICATION OF ABNORMAL LIPOPROTEIN ACCORDING
TO METABOLIC PATHWAYS:
Deficiency in lipoprotein lipase o Rare, autosomal recessive
activity disorder
o Deficiency of LPL is not a
predisposing factor to
atherosclerosis
o Laboratory pictures shows the
following: Marked with
hyperchylomicronemia and
hypertriglyceridemia; VLDL
normal; HDL is decreased; LDL
is increased
o Diagnosed in childhood
o Signs: abdominal pain; acute
pancreatitis
o If TAG reaches 2000 mg/dL=
eruptive xanthomas appear
o If TAG reachers 4000 mg/dL=
lipemia retinalis appears
Apolipoprotein C-II deficiency o Here, CMs do not break and
TAG level increases ( 500 to
10,000 mg/dL)
o Cholesterol level is high; HDL
and LDL values are very low
o Milder symptoms than
deficiency of LPL
o Occurs in older age
o Patients are not predisposed
to atherosclerosis
Familial combine hyperlipidemia o increased total and LDL
cholesterol; increased TAG; low
HDL
o common disease (1:100
individuals); expressed in
adolescence
o associated with coronary
heart disease
o symptom: atherosclerosis
Hyperapobetalipoproteinemia o Increased levels of apo B-100,
total cholesterol, TAGand LDL;
decreased HDL o High lipid is
due to overproduction of VLDL
in the liver
o associated with coronary
heart disease
o symptom: atherosclerosis
Familial hypertriglyceridemia o Normal LDL, decreased HDL,
increased TAG
Type V hyperlipoproteinemia
Dysbetalipoproteinemia (type
III)
Familial hypercholesterolemia
Familial
hypoalphalipoproteinemia
o Has an autosomal dominant
inheritance pattern with delayed
expression
o Common disease (1:500
individuals)
o VLDL is large
o Symptom: acute pancreatitis (
if TAG reaches 4,000 mg/dL)
o Increased CMs and VLDL
o Has an autosomal dominant
inheritance pattern
o Expressed in adulthood
o Symptoms: eruptive
xanthomas, lipemia retinalis,
pancreatitis, and abnormal
glucose tolerance with
hyperinsulinemia
o Not linked to premature
atherosclerosis
o Increased total cholesterol
and TAG; decreased LDL and
HDL
o Caused by a primary genetic
defect in the removal of CMs
and VLDL remnants
o Occurs in 1:1000 individuals
o Expressed in adulthood
o Symptoms: palmar
xanthomas, eruptive xanthomas
o Premature atherosclerosis
appears in 30 to 50% of
patients
o 2 to 3 times increased LDL;
increased T cholesterol and
TAGs; decreased HDL
o Occurs in 1:500 individuals o
Inherited in an autosomal
dominant pattern
o Caused by genetic mutation in
the LDL-receptor gene
o Cholesterol deposits in skin,
tendon, arteries
o Yellow-orange cutaneous
xanthomas appear before the
age 30
o Homozygous die before 40
years old from MI
o Normal TAG, normal LDL,
normal VLDL; decreased HDL
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o Inherited in an autosomal risk factor
dominant pattern ➔ LDL-C concentration (≥130 mg/dL; ≥3.4 mmol/L) with ≤2
o High incidence of CHD risk factors
Defects in the synthesis of o Normal cholesterol, normal ➔ LDL-C concentration (≥100 mg/dL; ≥2.6 mmol/L) with
apolipoprotein A-I TAG, normal LDL, Normal VLDL, CHD or risk equivalent
decreased HDL ➔ HDL-cholesterol (<40 mg/dL; <1 mmol/L)
o Heterozygous have half the ◆ Among the cholesterol mentioned, it is only
normal HDL value and HDL that when decreased, is a positive risk
homozygous have only a trace factor
amounts ➔ DM= CHD risk equivalent
o Symptoms: corneal clouding; ➔ Metabolic syndrome ( multiple metabolic risk factors)
risk developing premature CHD
Defects in catabolism of o Decreased HDL; decreased B. Negative risk factors
apolipoprotein A-I (Tangier’s Tcholesterol, normal LDL and ➔ When HDL is elevated and when the LDL id decreased
disease) VLDL, normal TAG ➔ HDL-cholesterol (≥60 mg/dL; ≥1.6 mmol/L)
o An inherited dominant pattern ● Since it a good cholesterol, it must be elevated.
o Accumulation of cholesterol ➔ LDL-C concentration <100 mg/dL; <2.6 mmol/L)
esters in body tissue ● Since it is a bad cholesterol, it must be
o Symptoms: hyperplastic decreased.
orange tonsils, splenomegaly,
peripheral neuropathy,
hepatomegaly, corneal clouding
o Homozygotes show increased
incidence of CHD
● The goal of the National Cholesterol Education Program
(NCEP) is to educate the public about the benefits of
lowering cholesterol levels to reduce the risk of CHD.
The 1992 pediatric guidelines for dyslipidemia have 2
approaches:
○ Diet and lifestyle changes
○ Targeted lipid screening for children with a
family history of heart disease and other
dyslipidemia cases

Risk factors in developing coronary heart disease –adults

(NCEP)

A. Positive risk factors

➔ Factors that would enhance the development of CHD
➔ Age: male ≥45 yrs old; female ≥ years old or premature
menopause
➔ without estrogen therapy
➔ Family history of premature CHD ( death due to MI
before 55 years of father or other female first degree
relative)
➔ Current cigarette smoking
➔ Hypertension (≥ 140/90 mmHg or anti-hypertensive
medication) Measurement of Blood Lipids
➔ LDL-C concentration (≥160 mg/dL; ≥4.1 mmol/L) with ≤1 ● The following guidelines are necessary before blood

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lipid is measured. specifically measures cholesterol and does not measure
1. Fasting: The ideal fasting time for lipid measurement is related sterols. This method is in agreement with the
12-14 hours. If patient can not tolerate, 9 hours maybe gold standard method ( USA-NIH), the so-called
accepted, however, this is considered as the minimum definitive method using isotope dilution mass
because the body clears hyperglyceredemia only 8 hours spectrometry.
after a meal. ❖ Salkowski or Zak reaction: Sulfuric acid and ferric ions
2. Position: Change in patient position results in a ~10% are used to produce a more stable red to reddish violet
decrease in total cholesterol, LDL-cholesterol, cholestadienyl disulfonic acid
HDL-cholesterol, apo A-1 and Apo B. A 50% decrease in
TAG is also observed.
3. Tourniquet: prolonged application of tourniquet will
increase the lipid concentration
4. Sample: serum sample is preferred for cholesterol, TAG
and HDLcholesterol by chemical methods. Plasma is
preferred for lipoprotein measurement by
ultracentrifugation or electrophoresis.
5. Anticoagulant. EDTA plasma was the traditional choice
for lipoprotein separation because it was thought to
enhance stability by chelating metal ions. However,
studies have shown that microclots formed in EDTA
unseparated from cells during storage can plug sample
probes. EDTA also osmotically draws water from RBCs,
diluting the plasma constituents. NCEP recommended
cholesterol measurements made on EDTA plasma
require correction by factor 1.03. B. enzymatic method
6. Storing: -20˚C for shorter storage and 70˚C or lower for ● CHOD-PAP. The cholesterol oxidase reaction produces
long-term storage hydrogen peroxide which is then used for Trinder
reaction. The final product is quinine-immine dye that is
Cholesterol Measurement spectrophotometrically measured at 500 nm.
➔ Cholesterol is preferably done on a 12-hour fasting
serum but studies show there is no significant change
after a meal. Specimen can be preserved at 4⁰C for
several days.
A. non-enzymatic method
● Non-enzymatic method involves the following general
steps: The absorbance reading is at 410 nm. ● Common Interferences: vitamin C and bilirubin interfere
1. extraction: this is needed to extract the cholesterol from with peroxidasecatalyze color reaction
lipoproteins Extraction is done using:
❖ Bloor’s reagent ( 3:1 ratio of ethanol and ether) Triglyceride Measurement
❖ Zeolite ➔ It involves the general steps:
2. saponification: KOH is used to hydrolyze cholesteryl esters so 1. extraction
that interference is avoided when reading free cholesterol 2. hydrolysis of TAG
3. purification: Free cholesterol is precipitated with digitonin. o enzymatic
4. color development: o alkali treatment
❖ Leibermann-Burchardt reaction: makes use of sulfuric 3. Measurement of the glycerol liberated
acid and acetic anhydride to produce an unstable green o Colorimetric
cholestadienyl monosulfonic acid. Color be stabilized by o Spectrophotometric
sodium sulfate. This used to be the reference method. o Fluorometric
❖ The current reference method (CDC) is GC-MS which ➔ Non-enzymatic method is the reference method but

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tedious and use only for lipid standardization laboratory
at the Centers for Disease Control and Prevention (CDC)
This involves these steps:
● Extraction is done using methanol, ethanol,
isopropanol, chloroformethanol (Folch’s reagent), or
diethyl ether (solvent of choice but hazardous)
● Lipoproteins are denatured to liberate the TAG
● Removal of interfering substances using:
○ Solvent partition using solvents ( ex. Nonane
and hexane)
○ Use of adsorbents
○ Use of sample blank- this is to correct for the
free glycerol ( this
○ should not undergo the hydrolysis step
➔ The hydrolysis of TAG to yield fatty acid and glycerol is
done by saponification. The methods are:
1. Treatment with alcoholic KOH at elevated
temperature. KOH is said to promote hydrolysis
while ethanol allows material to remain in the
solution and minimize reforming of TAG. The
increased temperature enhances the rate of
hydrolysis.
2. alcoholic trans-esterification
➔ The glycerol liberated is then oxidized to formaldehyde
using periodate. The formaldehyde is then measured
quantitatively using:
1. Eegrine’s reaction: uses chromotropic acid and sulfuric
acid to produce a pink end product that is measured at
565 nm.
2. Schryver’s reaction: uses phenyl
hydrazine/ferricyanide/HCl mixture to derive at an end
color that is measured at 540 nm.
3. Pay’s reaction: uses methyl benzothiazolinone/ferric
chloride mixture to produce a product that is read at 620
nm
4. Hantzsch reaction: uses ammonium acetate and acetyl
acetone to produce yellow diacetyl dihydrolutidine that
is measured at 410 nm. This is the method of choice for
measuring formaldehyde.
Enzymatic method
➔ Enzymatic method is done in one-step fashion. The
extraction step is eliminated.
1. Baculo-David (NADH consumption method)
2. Megraw (Formazan formation)
3. Winartasaputra
4. Nagele-Trinder reaction
➔ The enzymatic TAG reaction sequences react with any
endogenous free glycerol ( contributes to 10 to 20 mg/dL
overestimation). This is corrected by reagents used for
blanking, categorized as:
a. Double cuvette blank=accomplished with a
second parallel measurement using the TAG
reagent without the lipase to quantify free
glycerol. The glycerol blank measurement is
subtracted from the total glycerol measurement
obtained with the complete reagent to
determine a net or blank-corrected TAG result.
b. Single cuvette blank = begins also with a lipase
free reagent. A blank reading is taken to
measure endogenous free glycerol. Lipase
enzyme is added as a second reagent and
reading is again done.
c. -MS for TAG measurement is currently used by
CDC . This involves the hydrolysis of fatty acids
on TAG and the measurement of glycerol.
HDL-Cholesterol Measurement
➔ For routine diagnostic reason, HDL can be performed
using:
a. chemical precipitation:
- early time: use of centrifugation forces (1500 x gravity)
and lengthy centrifugation time ( 10 to 30 minutes)
- modern time: involves addition of a precipitating agent
to serum or plasma to aggregate non-lipoprotein then
centrifugation using high-speed centrifuge ( forces of
10,000 to 15,000 x gravity) with a decreased time ( 3 to
5 minutes)
Common precipitating agents used:
○ heparin with manganese= earliest method; to
precipitate apo-B
○ sodium phosphotungstate with magnesium-
developed because manganese interferes with
enzymatic assay. This is sensitive but with
greater variability
○ dextran sulfate (synthetic heparin) with
magnesium- more specific
○ polyethylene glycol- requires higher
concentrations of reagent leading to difficulty in
pipeting because the resulting product is more
viscous.
b. measurement of HDL in the supernate with analysis
applied to total cholesterol and quantified using a
spectrophotometer
c. 3-step procedure involving ultracentrifugation to remove
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VLDL; heparin manganese precipitation to remove LDL; A. Friedewald equation:
and analysis of supernatant by Abell-Kendall assay. ( VLDL– cholesterol = plasma TAG
this is the accepted reference HDL –chol measurement ( in mg/dL) 5
by CDC)
d. Because letter c is tedious and expensive, CDC uses VLDL-cholesterol = plasma TAG
direct dextran sulfate precipitation with Abell-Kendall ( in mmol/L) 2.175
cholesterol assay.
Then: LDL-cholesterol
Problem with precipitation: = total cholesterol- HDL cholesterol –VLDL cholesterol
➔ Interferences of HDL values is High TAG= prevents
sedimentation. The result is cloudiness, turbidity or B. Delong Equation:
particulate floating in the supernate. This will ead into VLDL– cholesterol = plasma TAG
overestimation of HDL-C. ( in mg/dL) 6.5

Improvement: VLDL-cholesterol = plasma TAG

➔ Development of homogenous methods which automate
the HDL quantification. However, the methods showed
lack of specificity for HDL measurements of patients
with liver or kidney conditions. Also, reagents are
constantly changed. This is the reason that
precipitation-based methods are still used by some
reference and research laboratories.
LDL methods
➔ The most common research method and the basis for
the reference method is beta quantification. This method
combines ultracentrifugation and chemical precipitation.
● Ultracentrifugation of serum at the native
density of 1.006 g/mL is used to float VLDL and
other CMs.
● Chemical precipitation is used to separate HDL
from the serum or from the infranate obtained
from ultracentrifugation.
● Cholesterol is also quantified in the serum, in
the 1.006 g/mL infranate, and in the HDL
supernate by enzymatic or other methods
● LDL is then calculated
● infranatant cholesterol-HDL cholesterol =
LDL-cholesterol
● total cholesterol – infranant cholesterol= VLDL-
cholesterol
If ultracentrifugation is not available:
1. measure TAG, total cholesterol and HDL cholesterol directly.
2. use the Friedewald or the Delong equation to find out the value
of other lipoprotein
( in mmol/L) 2.825
Then: LDL-cholesterol
= total cholesterol- HDL cholesterol – VLDL cholesterol
➔ The above formula works well in normolipemic
specimens. The presence of elevated TAG (400 mg/dL is
the accepted limit), CMs, and β-VLDL characteristic of
the rare type III hyperlipoproteinemia precludes the
Friedewald and Delong estimation.
➔ POCT now available for measuring lipids and glucose
simultaneously from a fingerprick sample
Apolipoprotein measurement
➔ Immunoassay to measure apo B, Lp (a)
➔ Agarose electrophoresis to measure Lp (a)
➔ Turbidimetry/ nephelometry to measure apo B and Lp (a)
Fatty acid measurement
➔ Enzymatic analysis using enzymes phospholipase D,
choline oxidase, and horseradish peroxidase.
Lipoproteins and clinical vascular disease
➔ Research showed that there is a strong correlation
between lipoprotein levels with atherogenesis. Lowering
LDL and increasing HDL values lowers the risk of
coronary atherogenesis.
Atherosclerosis
➔ It begins when atheroma is formed after an endothelial
cell damage.
➔ Damage of endothelial cells allows lipid deposition in

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the area.
➔ Macrophages detect the lipids, enter the site and
phagocytize the lipids.
➔ Macrophages cannot phagocytize all LDL, and some LDL
ends up accumulating the damaged blood vessels. The
activity of macrophages stimulate collagen formation
and the macrophage/LDL build up becomes fibrous.
➔ The build-up can weaken the vessel wall and lead to
thrombosis.
➔ This process repeats itself as young atheromas form.
Chronic scarring and inflammation with calcium
deposition in the site hardens the atheromas, they are
calcified.
Vasculitis
➔ Inflammation of arteries, rarely veins.
➔ Mostly autoimmune
➔ Can affect any organ of the body. Sequelae includes
aneurysm, stroke, gangrene, renal disease &
disseminated intravascular coagulation (DIC).
Additional notes:
Digestive phase
When the carbo would enter the intestine = neutralized, and
acted by other amylase released from the pancreas
(Amyloxine) is acting on the carbo and not on the lipids

● Risk factors: age, geneder, genetics, high cholesterol, Bile

hypetension, smoking, diabetes, high hemocysteine level - Stored in gallbladder
● Coronary atheromas cause ischemia (decreased blood - Released in liver
flow) and angina (chest pain). - Associated with the fats
● Atheromas blocking coronary vessel= MI; atheroma in - Role: emulsifying agent
aorta= weakens artery walls and allows bulges Exogenous pathway = Chylomicron
(aneurysm); atheroma in brain=brain infarction or stokes; Endo = VLDL
atheromas in lower extremeties= gangrene
When insulin is present and is active in its work do not expect
glycolysis to occur
Hypertension
When it is only cholesterol = no need to fast
TAG and LDL cholesterol measurement = atleast 12 hours of
fasting
- Rationale: to get rid of exogenous TAG

National cholesterol education program

- Educate the public why we should (hay ambot ano
nato)

EDTA - the specimen is plasma

- Correct by multiplying it to factor 1.03
➔ Sympathetic Nervous System (SNS) maintains healthy
blood pressure and tissue perfusion. Hyperactivity of
General steps
SNS leads to increased heart rate and peripheral
- Extraction
resistance due to blood vessel vasoconstriction.
- Hydrolysis of TAG
➔ Vasoconstriction stimulates renin release, increases
- Enzymatic
tubular sodium reabsorption, and reduces renal flow.
- Alkali treatment
These effects to increasing blood pressure.
- Measurement
- Photometry
➔ Many individuals with hypertension already have
Why KOH?
atherosclerosis, including coronary artery disease, thus,
- It would promote hydrolysis
they are more vulnerable to MI, cerebrovascular
- Why at high temp?
accidents, and heart failure. Kidneys are also affected
- Enhance the hydrolysis
(nephrosclerosis)

Enzymatic method = no extraction

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Proteins have great diagnostic significance because of their

Lipid assay vital functions:
● Cholesterol
● TAG 1. They make up enzymes that act as biological
● HDL catalysts.
● LDL 2. They also serve as transport and storage like
albumin, lipoproteins, hemoglobin, transferrin, and
If the TAG is 400mg/dL and above do not compute for the ceruloplasmin.
LDL 3. Plasma proteins are important in the distribution of
- Subject for ultracentrifugation water among water compartments in the body (
- LDL is falsely elevated decrease proteins= decrease colloidal osmotic
- Answer: cannot be computed pressure=increased interstitial fluid=edema)
- Ex: 4. The amphoteric nature of proteins provides the
- Compute VLDL mechanism for their action as buffers.
- LDL value cannot be computed due to high 5. Proteins participate in hemostasis ( coagulation of
TAG value blood)
Positive = lead to coronary heart disease 6. Several proteins are glycoproteins and as such they
Negative = are able to function which cells are native and which
Steatorrhea = fats in stool are foreign to the body.
References for LDL, VLDL, and total cholesterol 7. Actin and Myosin are contractile proteins for muscle
= same for male and female contraction.
Reference range for HDL 8. Immunoglobulins are proteins that protect the body
= slightly higher for female from microorganisms.
9. Some hormones like GH and ACTH are proteins that
have a crucial role in the function of the body system.
ADULT REFERENCE RANGE FOR LIPIDS 10. Many cellular proteins also act as receptors to
hormones
● TOTAL CHOLESTEROL: 11. Plasma proteins are important in tissue nutrition,
→ 140-200 mg/dL (3.6-5.2 mmol/L) providing up to 20% of the total required daily energy
● HDL-C by the body.
→ 40-75 mg/dL (1.0-2.0 mmol/L) 12. Proteins have a structural role. Collagen is the major
● LDL-C fibrous element of the skin, bone, tendon, cartilage,
→ 50-130mg/dL (1.3-3.4 mmol/L) blood vessels, and teeth. Elastin and proteoglycans
● TAG are connective tissue proteins.
→ 60-150 mg/dL (0.7-1.7 mmol/L)
➔ Proteins are made up of amino acids. Amino acids are
AMINO ACIDS AND PROTEINS structural units or building blocks of a protein. They are
➔ Proteins are the most abundant compounds in the body. bonded together by a peptide bond in a head to tail fashion
➔ Protein is a polymer of amino acids. (the carboxyl group of 1 amino acid links to the amino group
◆ Amino acids are structural units or building of another amino acid). 2 amino acids joined together are
blocks of protein. called dipeptide; 3 amino acids are called tripeptide; 4
➔ They are found in all cells of the body, as well as in fluids, together are called tetrapeptide; and as the chain increases,
secretions, and excretions. it is called a polypeptide. There are only 20 amino acids
➔ Proteins consist of carbon, oxygen, hydrogen, nitrogen, and usually found in a protein.
sulfur. It is nitrogen that sets protein apart from other
macromolecules.
➔ A typical protein consists of 200 to 300 amino acids, but
there are some which are smaller or larger.

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◆ Positively charged R groups
◆ Negatively charged R groups

➔ Amino acids are composed of amine group (N), acid group,

and the side chain.

➔ Among the 20 amino acids, there are 10 essential and the 9

are considered essential for the adults. Arginine is now
considered nonessential for adults.
➔ Out of the 20, there are 10 which are considered essential
because they are not produced in the body. Amino acids
➔ Deamination is the removal of amine group
which are synthesized in the body are called nonessential
➔ Transamination is the transfer of amino group
amino acids.
➔ Essential - it means they should be initially present in the
diet because the body cannot manufacture them.
➔ Both essential and nonessential have the same function.
The difference is that essential amino acids are not
produced in the body. Thus, it should be present in our diet.

14.0
➔ The resultant is called a keto acid which could enter into the
common metabolic pathway. For example in the citric acid
cycle, there are different amino acids that are entering into
it.
➔ Amino acids could be either Ketogenic or Glucogenic so
that it can result in the formation of energy when the body
has insufficient glucose supply.
➔ As a result of Deamination and Transamination, ammonium
➔ These 10 amino acids are recalled by using a mnemonic
ions are produced. This would be converted into a less toxic
code: PVT TIM HALL to mean phenylalanine, valine,
form in the urea cycle which happens in the liver.
threonine, tryptophan, isoleucine, methionine, histidine,
➔ There are 20 standard amino acids usually found in a
arginine, leucine, and lysine. Arginine is considered
protein and are categorized into 5:
semi-essential because it is already produced in an adult
◆ Nonpolar, aliphatic R groups
body.
◆ Polar, uncharged R groups
◆ Aromatic R groups

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➔ Some essential amino acids serve as precursors of
nonessential; thus, need to be essentially present in our
diet. For example, tyrosine (a nonessential amino acid) is
produced from phenylalanine (an essential amino acid).
➔ Amino acid general structure contains an amino group and a
carboxyl group bonded to the α-carbon. Amino acids differ
from one another by their R group.
➔ In some classifications, glycine is placed under non-polar
because it does not have a polar side chain or classified
under polar classification because it lacks nonpolar side
chain.
➔ Amino acids which are acidic have additional carboxyl
groups in their side chain. They are negatively charged at
neutral pH.
➔ Amino acids with basic side chains are positively charged at
or near neutral pH.

Table 2. Functions of Amino Acids

Essential AA Functions

Arginine (Arg) Cell division; wound healing; stimulation of

➔ There are 4 classifications of proteins. They are group 1 – protein synthesis; immune function; release of
amino acids with nonpolar side chains; group 2- amino acids hormones; conversion of ammonia into urea.
with electrically neutral polar side chains; group 3- amino ➔ It is also important for the synthesis of
acids with carboxyl groups in their side chains; and group creatinine.
4-amino acids with basic side chains. The succeeding table ➔ Can be obtained thru nuts, seeds, meat
classifies the 20 amino acids. products, meat products, and seaweeds.

Histidine (His) ➔ To help grow and repair body tissues;

Table 1. Categories of 20 Amino Acids
maintain the myelin sheaths that protect
nerve cells
Non-polar Polar Acidic Basic
➔ Direct precursor of histamine
(hydrophobic) (uncharged)
➔ Manufacturing of red & white blood cells
Leucine (leu,L) Glycine (Gly, G) Aspartic acid Lysine (lys, K) ➔ Protects the body from heavy metal
(Asp, D) poisoning
➔ Important source of carbon atoms in the
Proline (Pro,P) Serine (Ser,S) Glutamic acid Arginine (Arg, R
synthesis of purines for DNA and RNA
(Glu, E)
synthesis
Alanine (Ala, A) Threonine (Thr, T) Histidine (His, H) ➔ Helps grow and repair body tissues,
maintain myelin sheaths.
Valine (Val, V) Cysteine (Cys, C) ➔ Can be obtained thru meat, fish, poultry,
nuts, seeds, and whole grains.
Methionine (Met, Tyrosine (Tyr, Y)
M)

Tryptophan (Trp, Glutamine (Gln,Q)

W)

Phenylalanine Asparagines (Asn,

(Phe,P) N)

Isoleucine (Ele,I)

Glycine (Gly, G)

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Isoleucine (Ile), ➔ Three important amino acids
Leucine (Leu), ➔ Referred to as branched-chain amino acids;
Valine (val) promote healing of muscle tissue, skin, &
bones; help regulate blood glucose
concentrations; maintain energy levels
➔ Isoleucine (Ile) has an important role in
Hemoglobin formation.
➔ It is also have the function to maintain,, heal,
and repair muscle, tissue skin, and bones.
➔ Utilized for hemoglobin formation
➔ May be obtained from fish, meat, poultry,
lentils, nuts, and seeds.
➔ Leucine (Leu) is needed for optimal growth
of infants
➔ For nitrogen balance of adults.
➔ 2nd most common amino acid next to
glycine
➔ Can be obtained in dairy, soy, beans, and
legumes.
➔ Valine (Val) - for muscle metabolism and
coordination
➔ Constitute fibrous protein in the body
➔ Tissue repair
➔ Maintain nitrogen balance.
➔ Val is for treatments of muscle, liver, and
gallbladder diseases; mental, & emotional
problems like insomnia and anxiety; liver and
gallbladder disease
➔ May be obtained through beef, chicken, pork,
fish, tofu, seeds, nuts, whole grains
➔ Leu & Val aid in maintaining the nitrogen
balance in adults
Lysine (Lys) ➔ Production of antibodies ; lowering of TAG
levels; absorption and conservation of
calcium; role in the formation of collagen
➔ For proper growth and bone
development in children
➔ Proper nitrogen balance in adults
➔ Can be obtained in meat, eggs, soy,
black beans.
Methionine ➔ Start codon
(Met) ➔ A source of sulfur ( required for normal
metabolism and growth); assist in fat
breakdown; helps lead and heavy metal
detoxification; helps diminish muscle
weakness; prevents brittle hair
➔ Contributes to synthesis of epinephrine,
choline.
Phenylalanine ➔ Promotes alertness and vitality; elevates
(Phe) mood; decreases pain; aids in memory and
learning; used to treat arthritis and
depression.
➔ Phe uses an active transport channel to
cross the blood-brain barrier and is used by
the brain to produce norepinephrine (NE)- a
neurotransmitter that transmits signals
between nerve cells.
➔ Phe in large amount interferes with the
production of serotonin
➔ Phe is the direct precursor of tyrosine
➔ Component for medications
➔ For enhancing memory and alertness
➔ Today it is regulated to avoid abuse
Threonine ➔ Formation of collagen; elastin, and tooth
(Thr) enamel; production of neurotransmitters
➔ Helps maintain proper protein balance
➔ Aids liver function, metabolism, and
assimilation
Tryptophan ➔ Opposing effect of Phy
(Trp) ➔ Sleep and relaxation
➔ Metabolic precursor for serotonin, melatonin
(nuerohomorne), niacin (vitamin)
➔ Natural relaxant that alleviates insomnia
➔ Treatment of migraine headaches, aids in
weight control by reducing appetite
➔ Helps control hyperacidity in children
Non-essential Functions
Amino Acids
Alanine (Ala) ➔ Formed from breakdown of DNA or the
dipeptides, anserine and carnosine and
glycolysis
➔ Transfers of nitrogen from peripheral tissues
to the liver for processing and excretion;
production of antibodies; reduces buildup of
toxic substances from muscle protein
breakdown
Asparagine ➔ Derived from aspartic acid and ATP
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(Asn) ➔ Transport of nitrogen; role in synthesis of
ammonia
➔ Primary role: conversion of one amino acid
into another via amination or transamination
➔ First isolated from asparagus juice
➔ 1st amino acid to be isolated
➔ Most abundant amino acid to transport
nitrogen
Aspartic (Asp) ➔ Formed from oxaloacetate
➔ Precursor for other amino acids
➔ Metabolite in the citric acid cycle and the
urea cycle
➔ Generation of glucose from non sugar
sources
Cysteine (Cys) ➔ Synthesized from methionine
➔ Can be essential to infants and elderly who
have metabolic or intestinal absorption
problems
➔ Toxic thus, absorbed during digestion of
cystine
Glutamic acid ➔ Synthesized from transamination of alanine
(Glu) and aspartic acid
➔ Serves as neurotransmitter, its dysregulation
linked to epileptic seizures; metabolism of
sugar and fats
➔ Transports potassium into spinal fluid
➔ Epileptic seizures
➔ Used as food additive, flavor enhancer
(umami) - in forms of MSG
Glutamine ➔ Synthesized from glutamic acid (Glutamic
(Gln)) acid + amino acid= glutamine)
➔ Renal maintenance of acid-base balance;
➔ Fuel for healthy digestive tract;
➔ The basis of building blocks for the synthesis
of RNA and DNA; source of cellular energy
➔ Aids in immune function; transports
ammonia to liver
➔ Treatment of serious illnesses, injury,
trauma, burns, cancer treatment related side
effects
➔ Wound healing in postoperative patients
➔ Used by athletes (e.g. weight lifters)
➔ Renal maintenance of acid-base balance
Glycine (Gly) ➔ Synthesized from serine
➔ Synthesis of nucleic acids, bile acids,
proteins, peptides, purines, ATP, porphyrins,
hemoglobin, glutathione, creatine, bile salts,
glucose, glycogen
➔ Detoxification of compounds; synthesis of
bile acids; an inhibitory neurotransmitter in
CNC; metal complexing agent; retards
muscle degeneration; improves glycogen
storage ; promotes healing
Proline (Pro) ➔ Synthesized from glutamine; precursor for
hydroxyproline (manufactured into collagen,
tendons, ligaments, & cardiac tissue
➔ Involved in wound healing (cartilage);
strengthening of joints, tendons, and cardiac
tissue; works with vitamin C to promote
healthy connective tissue.
Serine (Ser) ➔ Formed from 3-phosphoglycerate (an
intermediate in glycolysis)
➔ For proper metabolism of lipids and fatty
acids; role in synthetic pathways for
pyrimidines, purines, creatine, & porphyrins.
➔ Highly concentrated in all cell membranes;
component of the protective myelin sheaths
surrounding nerve fibers; aids in the
production of antibodies
Tyrosine (Tyr) ➔ Formed from phenylalanine; precursor of
adrenal and thyroid hormones
➔ Aids in the function of the adrenal glands;
thyroid and pituitary glands; acts as a mood
elevator (treatment for chronic fatigue,
narcolepsy, anxiety, depression, low sex
drive, allergies, and headaches)
➔ Diet pill: suppress appetite
Table 3. Newly Discovered Amino Acids
Amino Acid Characteristics
Selenocystein ● Not coded for directly in the genetic code
e (Sec) ● Encoded as UGA (stop codon)
● Like other amino acids, it has a specialized
transfer RNA (tRNA)
● Present in enzymes formate dehydrogenase,
glycine reductase, some hydrogenase
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➔ The amino group is removed from amino acids by either
Pyrrolysine ● 22nd naturally occurring genetically encoded
deamination or transamination. The ammonium ion
(Pyl) amino acid used by some prokaryotes and
produced during deamination is converted into urea via the
single-celled microorganisms in enzymes
urea cycle in the liver. The resultant keto acid can enter into
that are part of their methane-producing
a common metabolic pathway with carbohydrates and fats.
metabolism
Those amino acids that generate precursors of glucose are
● A lysine-derivative that is encoded by the
called glucogenic. Examples are alanine (deaminated to
UAG codon (stop codon)
pyruvate); arginine (converted to αketoglutarate); aspartate
● Not present in humans
(converted to oxaloacetate). Amino acids that are degraded
into acetyl CoA are called ketogenic. An example of this is
leucine. There are also amino acids that can be both
ketogenic and glucogenic because some of their carbon
atoms emerge in ketone precursors and others appear in
potential precursors of glucose.
➔ Aminoacidopathies are rare inherited disorders of amino
acids. The abnormalities exist either in the activity of a
specific enzyme in the metabolic pathway or in the
membrane transport system for the amino acid. Due to the
severity of complications of aminoacidopathy that newborn
screening tests become mandatory.

Phenylketonuria (PKU)
➔ Is an autosomal recessive inherited disease in 1:15,000
births in America.
➔ REMEMBER: enzymes and the problems occuring in the
diseases
➔ There is the absence of phenylalanine hydroxylase (PAH is
an enzyme that catalyzes the conversion of phenylalanine to
tyrosine).
◆ Upper normal limit of blood phenylalanine for full
term normal weight infants range is 2 mg/dL (120
µmol/L)
◆ In the absence of PAH, phenylalanine can reach
more than 1200 µmol/L
◆ Urine has a “musty” or “mousy” odor due phenyl
pyruvic acid and other metabolites.
◆ Chronically elevated levels of phenylalanine and its
metabolites can cause significant and permanent
brain damage.
◆ Brain damage is avoided if the disease is detected at
Amino Acid Metabolism birth and the baby is maintained on a low
➔ Proteins are digested by proteolytic enzymes, pepsin, and phenylalanine diet. Women with untreated PKU
trypsin into amino acids. The amino acids are then rapidly during pregnancy almost always have microcephalic
absorbed from the intestine into the portal blood and and mentally retarded babies.
subsequently become part of the body's pool of amino acids. ◆ Hyperphenylalaninemia can also result from a
The amino acid pool is used for the synthesis of proteins and deficiency in the enzymes needed for the
non-protein nitrogen-containing compounds such as purines, regeneration and synthesis of tetrahydrobiopterin
pyrimidines, porphyrins, creatine, histamine, thyroxine, (BH4). This occurs only in 1-5% of cases.
epinephrine, and coenzyme NAD. ◆ BH4 is a cofactor required for the enzymatic

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hydroxylation of phenylalanine, tyrosine, and
tryptophan.
◆ Sapropterin dihydrochloride (kuvan)
● the first drug to manage PKU
→One component of newborn screening test
Tyrosinemia
➔ Is an inherited autosomal recessive disease. It is
➔ PKU is detected in the laboratory using serum, whole blood,
or urine samples:
1. Guthrie bacterial inhibition assay
● This is a semiquantitative test.
● Spores of Bacillus subtilis are incorporated into an
agar plate that contains ß2 –thienylalanine
(inhibitor) a metabolic antagonist to B. subtilis
growth.
● A filter paper disk impregnated with blood from the
infant is placed on the agar.
● High blood level of phenylalanine counteracts the
antagonist, and bacterial growth occurs.
● To avoid false negative results, the test must be
carried out on an infant of at least 3 days old to
ensure adequate time for enzyme and amino acid
levels to develop.
● The sample must also be collected before any
administration of antibiotics or transfusion.
● This test is sensitive enough to detect phenylalanine
concentrations of 3 mg/dL (180 µmol/L).
● This test has been replaced by HPLC ( the reference
method) and MS/MS (the gold standard for
detecting 25 congenital diseases using a single
specimen in newborns).
Reasons of False (-) Guthrie result
➔ infant not being at least 24 hrs
➔ transfusion of blood products
➔ when the specimen is taken after antibiotic treatment
2. Microfluorometric assay
● This is a quantitative direct measurement of
phenylalanine in dried blood filter discs.
● This test is more adaptable to automation and is not
affected by antibiotics.
● The test requires pretreatment of dried blood filter
test with trichloroacetic acid (TCA).
● The extract is then added in a microtiter plate to a
mixture of ninhydrin, succinate, and leucylalanine in
the presence of copper tartrate.
● The fluorescence of the complex is measured using
excitation/emission wavelengths of 360 and 530 nm
respectively.
characterized by the excretion of tyrosine and its catabolites
in the urine (tyrosinuria). The 3 types are:
1. Type 1 tyrosinemia
● Most Severe form
● which occurs in 1 in 100,000 births worldwide.
● Caused by mutation in the fumarylacetoacetate
hydrolase (FAH) gene
● FAH is the last of the 5 enzymes for the metabolism
of tyrosine
● Symptoms
○ manifest in the first few months of life to
include: failure to thrive, diarrhea, vomiting,
jaundice, a cabbage-like odor, distended
abdomen, swelling of legs, & increased
predisposition for bleeding
● This can lead to liver and kidney failure, problems
affecting the nervous system ( peripheral
neuropathy, and an increased risk of liver cancer
later in life)
● Untreated patients do not usually live past 10 years
old
2. Type II tyrosinemia
● caused by mutation in the
tyrosineaminotransferase (TAT) gene
● TAT is the first enzyme in tyrosine metabolism
● Occurs in less than 1 in 250,000 births worldwide.
● ½ of patients have impaired mental development
● Other symptoms:
○ photophobia, eye pain and redness,
painful skin lesions on the palms and
soles of the feet
3. Type III tyrosinemia
● A rare disorder
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● Caused by mutation in 4-hydroxyphenylpyruvate
dioxygenase (HPD) gene o HPD is the second
enzyme in the tyrosine metabolism
● Symptoms:
○ impaired mental development, seizures,
some intermittent loss of balance and
coordination.
● Diagnostic criteria for tyrosinemia: elevated tyrosine
in the blood detected using MS/MS. There is also
high succinylacetone
● Succinylacetone causes damage to the liver and
kidneys
● Treatment:
○ low-protein diet; medication Nitisinone that
has replaced liver transplantation
Alkaptonuria
➔ Alkaptonuria is also an inherited autosomal recessive
disease. It occurs in 1 in 250,000 births worldwide
◆ Caused by mutation in the homogentisate oxidase
(HGD) gene
◆ The predominant manifestation of the disorder is
the change of urine color into brownish-black
because urine HGD when exposed to air will
oxidize.
◆ Patients have no immediate problems until late
stage of disease when HGA accumulates in the
connective tissues (cartilage of the nose, ears,
tendons of extremities) causing dark blue-black
pigmentation of these tissues (ochronosis). Small
dark spots in the sclera can also be observed
◆ Patients also experience an arthritis-like
degeneration ( as HGA deposits in the cartilage of
joints like the hips)
◆ Detected using a rapid screening test, ferric chloride
test. Ferric chloride + HGA = black color due to the
interaction with the phenol group
◆ Treatment: high dose of vitamin C to slow the
progression of arthritis
Maple Syrup urine disease (MSUD)
➔ MSUD is a hereditary disease in an autosomal recessive
pattern that results from an absence or reduced activity of
branched-chain keto acid decarboxylase (BCKD), thereby
blocking the normal metabolism of branched-chain amino
acids leucine, isoleucine, and valine.
➔ This results to the accumulation of the 3 branched-chain
amino acids and their corresponding keto acids in the blood,
urine, and CSF.
➔ The striking feature of the disease is the maple or burnt
sugar odor of the urine, breath, and skin.
➔ Occurs in 1 in 185,000 births worldwide
➔ Infants with MSUD appear normal until 4 to 7 days old.
They develop lethargy, vomiting, and signs of failure to
thrive. CNS symptoms include muscle rigidity and stupor
and respiratory irregularities. If untreated, the disease
causes severe mental retardation, convulsions, acidosis, and
hypoglycemia.
➔ Controlled by restricting dietary proteins to minimize
leucine, isoleucine, and valine
➔ Laboratory tests for MSUD include:
1. Modified Guthrie Test
● The metabolic inhibitor to B. subtilis included in
the growth media is 4-azaleucine
2. Microfluorometric assay
● Is used for massive screening
● This uses a mixture of methanol and acetone to
denature the hemoglobin.
● Enzyme leucine dehydrogenase is added to the
aliquot and the fluorescence measured at 450
nm. Level of leucine at 4 mg/dL= presence of
MSUD
3. MS/MS
● Is also used for screening and quantitative
analysis
4. Prenatal diagnosis for MSUD
● Is made by testing the decarboxylase
concentration of amniotic fluid
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Homocystinuria
Isovaleric Acidemia (IVA)
➔ It is an autosomal recessive disorder arising from mutation
of IVD gene resulting in deficiency of isovaleryl-CoA
dehydrogenase in the degradative pathway of leucine.
◆ Occurs in 1 in 250,000 births in the USA o Patients
of IVA can be symptomatic and asymptomatic
◆ Typical characteristic of the condition is the “sweaty
feet” odor.
◆ Other features occurring as early as few days after
birth: failure to thrive, vomiting, lethargy o Health
issues related to IVA can be mild to severe, such as
seizures and coma, or permanent damage to the
brain and nervous system and possible death
◆ Treatment: protein-restrictive diet to prevent
accumulation of isovaleric acid ( toxic to CNS); oral
administration of glycine and carnitine
◆ Laboratory test: urine of newborns screened for IVA
using MSMS or chromatography technique.
◆ IVA patient laboratory results: metabolic acidosis,
mild to moderate ketonuria, hyperammonemia,
thrombocytopenia, neutropenia
➔ It is also an inherited autosomal recessive disorder caused
by mutation of CBS gene that encodes for cystathionine
ß-synthase which results to a plasma and urine elevation of
homocysteine and methionine precursors The enzyme
requires vit b6 as a cofactor).
◆ Occurs in 1 in 200,000 births worldwide.
◆ Infants show no defect but the symptoms appear
gradually with age.
◆ Associated clinical findings include thrombosis due
to toxicity of homocysteine to the vascular
endothelium, osteoporosis, dislocated lenses in the
eye due to the lack of cysteine synthesis essential
for collagen formation, and frequently mental
retardation.
◆ Treatment: dietary protein restriction; high doses of
B6
◆ If patient does not respond to mentioned treatment:
supplementation of trimethylglycine and folic acid
➔ Neonatal screening can be done through:
1. Guthrie test using L-methionine sulfoximine as the
bacterial growth inhibitor.
2. HPLC & MS/MS confirm positive results of
screening test( methionine greater than 2 mg/dL=
hemocystinuria
3. LC-MS/MS is used for urinary total hemocysteine
test
4. Cyanide-nitroprusside spot test. A urinary test for
the measurement of homocystine ( red-purple
color). Because cysteine can also react with cyanide,
the presence of homocystine is confirmed using
silvernitroprusside test. ( reddish color).
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Citrullinemia
➔ This genetic disease belongs to urea cycle disorders. It is an
inherited autosomal recessive pattern condition.
◆ Treatment: restriction of high caloric protein caloric
diet; arginine supplementational administration of
sodium benzoate and sodium phenylacetate
◆ There are 2 forms:
1. Type 1 citrullinemia
● Most common: occurring in 1 in 57,000 births
worldwide
● Result of a mutation in the argininosuccinic acid
synthase (ASS I) gene
● Accumulation of citrulline and ammonia in the blood
● Infected infants show: lack of appetite, failure to
thrive, vomiting, lethargy, seizures, coma as toxic
ammonia accumulates. If not treated promptly can
result to severe brain damage and death
2. Type II citrullinemia
● Caused by mutation of SLC25A 13 gene that codes
for production of transport protein citrin.
● Citrin helps transport molecules inside the cell that
are used for the production and breakdown of
simple sugars, breakdown of proteins, and the urea
cycle
● Since type II citrullinemia does not have citrin,
ammonia and other toxic substances accumulate
leading to confusion, restlessness, memory loss,
personality changes, seizures, & coma
Arginosuccinic Aciduria
➔ This is also a urea cycle amino acid disorder, inherited in an
autosomal recessive pattern.
➔ This results from the mutation of Arginosuccinic acid lyase
(ASL) gene; the enzyme is important in the conversion of
ammonia into urea
➔ Symptoms develop within a few days of life beginning with
lethargy and unwillingness to eat.
➔ Treatment is similar to citrullinemia o Newborn screening
cannot differentiate arginosuccinic aciduria and citrullinemia
Cystinuria
➔ It is an inherited autosomal recessive disorder occurring in 1
in 10,000 births worldwide.
◆ There is a defect in SLC3A1 and SLC7A9 genes that
code for the synthesis of a protein complex found in
kidneys that is responsible for the reabsorption of
cystine from urine
◆ Problem: amino acid transport not the enzyme
◆ Cystine can precipitate and form stones in kidneys,
ureters, or bladder
◆ Treatment: increase to 4L/day patient’s fluid intake;
if fluid does not reduce stone formation tendencies,
Penicillamine is prescribed
◆ May require percutaneous nephrolithotomy (kidney
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stone removal) ◆ The tertiary structure refers to the way in which the
◆ Laboratory diagnosis: twisted chain folds back on it to form the
1. Cyanide-nitroprusside (red-purple color reaction with three-dimensional structure. The specific
sulfhydryl groups) convolutions a polypeptide undergoes are
2. Ion exchange chromatography is used for cystinuria determined by the interaction of the R groups in the
patients with elevated lysine, arginine, & ornithine molecule. The reactions of the R groups include
disulfide linkages, electrostatic attractions,
hydrogen bonds, hydrophobic interactions, and van
Amino Acid Analysis
der Waals forces. This structure is responsible for
many of the physical and chemical properties of the
1. Blood sample collected in heparin is collected 6-8 hour
protein.
fasting to avoid the effect of absorbed amino acids
◆ Quaternary structure is the arrangement of two or
originating from the dietary proteins.
more polypeptide chains to form a functional protein
2. The plasma is promptly separated from the cellular
molecule. An example of this structure is
elements in the manner not to aspirate the platelets and
hemoglobin. The polypeptide chains are joined by
leukocytes because these cells have 100 fold of aspartic
noncovalent bonds like hydrogen and electrostatic.
and glutamic acid than in the plasma. Hemolysis is also
avoided because of the same reason.
3. Tests should be carried immediately within 30 minutes of
sample collection or sample is preserved at -20˚C to
-40˚C.
4. Random urine samples can be used for screening amino
acids; a 24-hour urine specimen is required for
quantitation.
5. Amniotic fluid can also be also analyzed.
6. TLC is the method of choice for screening purposes

Protein Structure
➔ The conformation (shape) of a protein is determined by
interaction between a polypeptide and its aqueous
environment in which the polypeptide attains a stable
3-dimensional structure. There are 4 levels of protein
structure:
◆ The primary structure refers to the number, kinds,
and order or sequence of amino acids in the
polypeptide. It is stabilized by a covalent peptide
linkage. This protein structure is crucial for the
function and molecular characteristics of a protein.
Any change in the amino acid composition can alter
the protein. For example when valine is substituted
for glutamic acid in the ß chain of hgb A, hgb S is
formed. ➔ When protein structure is disturbed, proteins lose their
◆ The secondary structure is the winding of the functional and molecular characteristics. This is called
polypeptide chain. This structure is maintained by denaturation. Denaturation is brought by heat, hydrolysis by
hydrogen bonds between the NH and CO groups strong acid or alkali, enzymatic action, exposure to urea or
either within the same chain or between different other substances, or exposure to ultraviolet light.
chains within the same molecule.
3 Phases of Protein Metabolism
1. Gastric phase

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● In the stomach, protein is digested by pepsin. Pepsin
is an activated pepsinogen. Pepsinogen is activated
by gastric acidity (HCl) and gastrin. Gastrin is a
hormone produced by the antrum (distal end of the
stomach) during ingestion of food.
2. Pancreatic phase
● Once the chyme reaches the duodenum, the
exocrine pancreas releases the proteolytic enzymes
trypsin, chymotrypsin, elastase, ( these 3 are
endopeptidases) and carboxypeptidase ( an
exopeptidase). Endopeptidases cleave proteins in
their internal sites while carboxypeptidases cleave
one amino acid from carboxyl-terminus of the
polypeptide
● Secretin stimulates the pancreas to produce a Catabolism and Nitrogen Balance
protein-free electrolyte solution rich in carbonates. It ➔ Unlike fats and carbohydrates, proteins have no designated
is produced by the duodenum in response to chime. storage. Dietary intake helps in providing the essential
Bicarbonates are needed to neutralize the acidity of amino acids. On the other hand, most proteins in the body
the chyme. are repetitively manufactured and then degraded allowing
3. Intestinal phase for efficient recycling of amino acids. A balance exists
● This phase is mediated by peptidases produced by between anabolism (protein synthesis) and catabolism
the mucosal cells. Aminopeptidases and (protein breakdown).
dipeptidases hydrolye the residual peptides. The ◆ Nitrogen balance is maintained by equal intake and
end products are amino acids and short peptides. excretion of amino acids.
◆ Positive nitrogen balance means their nitrogen
intake exceeds their loss as net protein synthesis
proceeds. This is seen in pregnant women, growing
children, adults recovering from major illness
◆ Negative nitrogen balance occurs in conditions
where there is excessive tissue destruction like
burns, wasting disease, continual high fevers, or
starvation.

Plasma Proteins
➔ The liver forms most of the albumin as well as the alpha
and beta globulins. The reticuloendothelial system and
immature plasma cells in the spleen, lymph nodes, and
bone marrow produce gamma globulin.

Prealbumin (Transthyretin)
➔ The carbon skeleton (α-ketoacids) could be used to glucose
➔ Name as such because it migrates ahead of albumin in the
or glycogen or used to produced ketone bodies. All amino
electrophoretic field.
acids except leucine and lysine are glucogenic.
➔ It is rich in tryptophan and contains 0.5% carbohydrate.
➔ This protein is an indicator of poor protein nutrition status.
It binds thyroid hormones to serve as their transport
mechanism. It also binds with retinol-binding protein to
form a complex that transports retinol (vitamin A).
➔ The reference range in adults is 0.1-0.4 g/L. It has a half-life
of 2 days

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➔ Prealbumin is decreased in hepatic damage, acute phase
inflammatory response, and tissue necrosis.
➔ Prealbumin values are increased in patients receiving
steroids, in alcoholism, and in chronic renal failure.
Albumin
➔ This is the most abundant protein in the serum (54%).
➔ It performs important functions in the body: a) It is
responsible for nearly 80% of the colloid osmotic pressure
(COP) of the intravascular osmotic pressure of the plasma,
which in turn maintains normal water distribution to blood
volume; b) acts as buffer; c) bind ( it has 4 binding sites)
and transport various substances in the blood
➔ Among the molecules transported by albumin are
unconjugated bilirubin, fat soluble hormones such as
thyroxine, penicillin, cortisol, and estrogen; fatty acids.
These substances are insoluble in plasma therefore they
need transport proteins to render them soluble. Albumin
also carries calcium and magnesium ions, iron to prevent
their excretion through the kidneys.
➔ The capacity of albumin to bind to dyes is the basis for
many methods for its quantification. Examples of
dye-binding procedures are Bromcresol green, methyl
orange, and hydroxyazobenzene benzoic acid (HABA).
➔ Recent studies have focused on clinical application of
glycated or glycosylated albumin as a more sensitive
indicator of short-term hyperglycemic control than glycated
hemoglobin because of shorter life span of albumin which is
20 days as compared to HbA1C which is 120 days. Affinity
chromatographic methods are utilized.
➔ Albumin leaves the bloodstream at a rate of 4% to 5% of
the total intravascular albumin concentration per hour. The
rate of movement is known as transcapillary escape rate (
which measures systemic capillary efflux of albumin)
➔ Analbuminemia means a genetic absence of albumin in the
blood resulting in autosomal recessive trait.
➔ Bisalbuminemia is the presence albumin with an unusual
molecular characteristic. This albumin exhibits two albumin
bands instead of the usual single bands in electrophoresis.
➔ A decreased concentration of serum albumin maybe caused
by:
◆ Acute inflammatory response as albumin is negative
acute-phase reactant
◆ Inadequate source of amino acids, which is seen in
malnutrition and muscle wasting diseases.
◆ Liver disease resulting in the inability of hepatocytes
to synthesize albumin
◆ Gastrointestinal loss as interstitial fluid leaks out in
inflammation and disease of the intestinal mucosa. o
◆ Loss in the urine.
➔ An increased albumin is seen in dehydration because the
albumin is contacted in a reduced volume of serum.
Globulin
➔ Globulins account approximately 38% of plasma proteins
➔ It consists of α1,α2, ß and γ fractions. Each fraction has
specific functions:
◆ α1 –antitrypsin (ATT)
● Is an acute-phase reactant, thus, increased in
inflammatory conditions
● Main function is to inhibit protease like trypsin
and elastase. In the lungs, elastase is released
by the neutrophils in inflammatory conditions to
combat invading microorganisms but can also
destroy alveoli leading to emphysema if not
inhibited by ATT.
● Mutations in SERPINA1 gene can lead to
deficiency of ATT.
● Abnormal form of ATT can accumulate in the
liver causing cirrhosis
● Increased levels of ATT is seen in pregnancy
and contraceptive use.
● Laboratory picture: lost of α1 –globulin in
serum electrophoresis
● Quantitative confirmatory analyses: radial
immunodiffusion; automated
immunonephelometric assays
● Phenotyping maybe done using immunofixation
assays
◆ α1-Antichymotrypsin (α1-ACT)
● Is a serine proteinase with cathepsin G1
pancreatic elastase, mast cell chymase, and
chymotrypsin.
● Carries 4 oligosaccharide side chains and
migrates between the α1 and α2 zones.
● Mutations in α1-ACT are identified in
Parkinson’s disease and chronic obstructive
pulmonary disease; also associated in
Alzheimer’s disease
● Increased levels are seen in inflammation and
injury. Hereditary deficiency is associated with
asthma and liver disease.
◆ Inter - α1-Trypsin Inhibitor (ITI)
● Is made up of at least 3 polypeptide subunits (2
heavy chains, H1 and H2; 1 light chain L or
bikunin).
● The light chain is responsible for the inhibition
of the proteases trypsin, plasmin, and
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chymotrypsin.
● ITI migrates in the zone between α1and α2
fractions.
◆ Gc-globulin (group specific component: Vit
D-binding protein)
● Also migrates between the zones of α1and α2.
● Inhibits the binding affinity for vitamin D with
actin.
● Elevation is seen in the 3rd trimester of
pregnancy and in patients taking estrogen oral
contraceptives.
● Low level is associated with severe liver
damage and protein-losing syndromes.
◆ ß2 –Microglobulin (B2
● Is the light chain component of the major
histocompatibility complex (HLA).
● This protein is found in the surface of most
nucleated cells, being the highest in
lymphocytes.
● It is small, hence, can be filtered and
reabsorbed in the kidneys.
● Elevated level is due to impaired kidney
clearance or overproduction of proteins as seen
in inflammatory conditions like rheumatoid
arthritis and lupus erythematosus. B2 is high in
patients with HIV, an indication that a large
number of lymphocytes are attacked and killed
by the virus.
● Laboratory analyses: high resolution
electrophoresis but because of low
concentration, it is measured by immunoassay
◆ α1-Acid Glycoprotein (Orosomucoid)
● Comprises 5 carbohydrate units attached to a
polypeptide chain.
● Has a low pI (2.7) and is a negatively charged.
● Is involved in the formation of certain
membranes and fibers in association with
collagen and may inactivate progesterone.
increased in inflammation, tissue damage, AMI,
trauma, pregnancy, cancer, pneumonia,
rheumatoid arthritis, and other condition
associated with cell proliferation.
● Neonatal serum AAG is useful to determine
bacterial infection
● Can be analyzed using radial radial
immunodiffusion, immunoturbidity,
Nephelometry, and immunofixation
◆ α2- Macroglobulin (AMG)
● Is the largest non-immunoglobulin proteins in
the plasma.
● Synthesized in the liver and is the major
component of the α2 band in electrophoresis
● Inhibits proteases such as trypsin, thrombin,
kallikrein, and plasmin by means of a bait
region that can entrap proteinases. After
binding and inhibiting proteinases, α2-
macroglobulin is removed by the RES
● Increases 10 folds or more in nephrotic
syndrome; 20% increase during pregnancy
● Increases also seen in DM, liver disease, and
with use of contraceptives
● Laboratory analyses: radial immunodiffusion,
immunonehelometry, enzymelinked
immunosorbent assay (ELISA), and latex
agglutination
◆ α1-Fetoprotein (AFP)
● Synthesized initially by the fetal yolk sac and
then by the parenchymal cells of the liver.
● Decreases gradually from after birth, reaching
adult concentrations by 8 to 12 months
● Although the function of AFP is not well
established, the protein protects the fetus from
immunolytic attack by its mother.
● Elevated levels seen in spin bifida, neural tube
defects, abdominal wall defects, anencephaly,
general fetal distress, and the presence of twins
● Low level indicates risk for trisomy (Down
syndrome) and trisomy 18 (Edwards syndrome)
● AFP screening done between 15 to 20 weeks
of gestation. It is important to require accurate
dating of pregnancy and correct the variables (
weight of mother, race, & diabetes) that might
be affecting the test.
◆ Haptoglobin (α2 glycoprotein)
● Synthesized in the liver and RES.
● A positive acute-phase protein
● Has 2 kinds if chains: 2 α chains and one ß
chain.
● Primary function: binds free hemoglobin and
prevents hemoglobin and iron loss into the
urine.
● Used to evaluate and differentiate hemolytic
anemia from other types.
○ Hemolytic anemia: high reticulocyte, low
hematocrit, low hemoglobin, low RBC
count, low haptoglobulin:
○ Anemia due to destruction of RBCs by liver
and spleen: high reticulocyte, with normal
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haptoglobulin
○ Anemia not elated to RBCs breakdown:
normal haptoglobulin and reticulocyte
● Physiologically, increases with age with higher
value in male.
● Pathologically, increased level is seen in
inflammation, rheumatic disease burns and
nephritic syndrome. Decreased level is found in
HDN.
● Haptoglobulin phenotype is linked as
independent risk factor to CVD in patients with
type 2 DM
● Measurement is through radial immunoassay
and Nephelometry.
Ceruloplasmin
➔ Is a copper containing α2 glycoprotein that has enzymatic
activities
➔ A positive acute-phase reactant protein
➔ increases with age with female having a higher level
➔ Physiologic elevation is seen in pregnancy and use of
contraceptives.
➔ Abnormal increase is seen in inflammatory process and
malignancies.
➔ Abnormal low level is observed in Wilson’s disease
(hepatolenticular degeneration), an autosomal recessive
inherited disease.
➔ It is further observed that copper is deposited in the skin,
liver, and brain, resulting in degenerative cirrhosis and
neurologic damage. Copper may deposit in cornea, resulting
to Kayser-Fleischer rings.
➔ Low ceruloplasmin is seen in malnutrition, malabsorption,
severe liver disease, nephrotic syndrome, and Menkes’
Kinky-hair syndrome (a disease which features decrease
absorption of copper)
➔ Ceruloplasmin is measured using copper oxidase,
immunochemical methods like Nephelometry and radial
immunodiffusion.
Transferrin (Sideroplasmin)
➔ A negative acute-phase glycoprotein synthesized by the
liver
➔ A major component of the ß-globulin fraction on protein
electrophoresis
➔ The major roles of transferrin are transport of iron and the
prevention of loss of iron through the kidney.
➔ A decreased in transferrin level generally reflects an overall
decrease in protein synthesis as seen in liver disease or
malnutrition. Patients with hereditary transferrin
deficiencies show hypochromic anemia. An increase of iron
bound to transferrin is found in a hereditary disorder of iron
metabolism called hemochromatosis. This disorder is
associated with bronze skin, cirrhosis, DM, and low plasma
transferrin levels.
➔ This protein is measured using immunodiffusion and
immunonephelometry.
Hemopexin
➔ Acute phase reactant protein produced by the liver.
➔ Its function is to remove circulating heme ( free heme called
ferroprotoporphyrin lX) in a 1:1 ratio.
➔ It also removes ferriheme and porphyrins.
➔ The heme-hemopexin complex is carried and destroyed in
the liver.
➔ The protein is low at birth but reaches adult value at 1 year
old. The value is elevated at pregnancy, DM, Duchenne type
of muscular dystrophy, and malignancies like melanoma.
➔ It is decreased in hemolytic disorders, administration of
diphenylhydantoin.
➔ This protein migrates in ß-region and can be determined in
the sample by radial immunodiffusion.
Lipoproteins
➔ Are complexes of proteins and lipids.
➔ It transports cholesterol, triglycerides, and phospholipids in
the blood. In electrophoresis, HDL migrate between
albumin and α1-globulin zone; VLDL migrate at the
beginning of the ß-globulin fraction, LDL appear a separate
band in the ß-globulin region
Complement
➔ Refers to group of proteins that participate in immune
reaction and serve as a link to the inflammatory response.
➔ These proteins circulate in the body as nonfunctional
precursors.
➔ Complement C3 is the most abundant complement protein
in human serum, followed by C4
➔ In the classic pathway, activation of these proteins begins
when the first complement factor, C1q, which binds to an
antigen-antibody complex. Each complement protein
(C2-C9) is then activated sequentially and can bind to cell
membrane, leading to lysis of cell
➔ In Properdin (an alternate pathway), early components are
bypassed and the process begins with C3. This pathway
does not require the presence of an antibody but the lytic
attach on the cell membrane follows the same sequence of
C5 to C9
➔ Increased C3 and C4 are linked to acute inflammatory
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disease and inflammation
➔ Decreased C3 typically is associated with autoimmune
disease, neonatal respiratory distress syndrome,
bacteremia, tissue injury and chronic hepatitis
➔ Decreased C4 is associated with DIC, acute
glomerulonephritis, chronic hepatitis, SLE
➔ C3 is used as screening test because of its pivotal position
in both classic and alternative pathways; C3 is most
sensitive indicator for classic pathway and C4 is a sensitive
measure of mild classic pathway activation
➔ Laboratory analyses: nephelometric immunoassay and
turbidimetry
Fibrinogen
➔ Is a liver synthesized glycoprotein and is one of the largest
proteins in the plasma.
➔ It is a positive acute-phase reactants.
➔ It migrates as a separate band between ß- and γ-globulins.
➔ Fibrinogen is important for clot formation.
➔ Increases in pregnancy and use of oral contraceptives
➔ Decrease level is seen in extensive coagulation
➔ It is analyzed using Biuret, Kjeldahl. Level is increased in
pregnancy and use of oral contraceptives.
C-Reactive Protein (CRP)
➔ Is produced by the liver and is one of the first acute-phase
proteins to rise in inflammatory disease
➔ Previously it was known to precipitate with the C substance
( a polysaccharide of pnuemococci), thus, the reason why it
was given such name. Later findings reveal that this protein
increases sharply whenever there is tissue necrosis,
whether the damage comes pneumococcal or other sources.
This led to the discovery that CRP recognizes and binds to
molecular groups found on a wide variety of bacteria and
fungi.
➔ CRP bound to bacteria promotes the binding complement.
This facilitates their uptake by phagocytes. The process of
protein coating to enhance phagocytosis is called
opsonization.
➔ CRP bound to bacteria promotes the binding complement.
This facilitates their uptake by phagocytes. The process of
protein coating to enhance phagocytosis is called
opsonization.
➔ CRP is increased in acute rheumatic fever, bacterial
infections, myocardial infarction, rheumatoid arthritis,
carcinomatosis, gout, and viral infections.
➔ The American Heart Association and CDC issued a joint
statement correlating association of inflammatory markers
(mainly CRP) and coronary heart disease and stroke
➔ Other studies show high CRP in colon cancer, complications
of diabetes, obesity, & the risk of developing type 2 DM
➔ CRP is measured using immunologic methods like latex
precipitation test, Nephelometry, and enzyme immunoassay
(EIA)
High sensitivity CRP (hsCRP)
➔ Named for a monoclonal antibody-based test method that
can detect CRP lebels below 1 mg/L
➔ To determine risk of CVD= High levels can predict recurrent
coronary events in patients with unstable angina and AMI
Immunoglobulins (Igs)
➔ Are glycoprotein comprised of 2 long polypeptide chains
(heavy or H chain) and 2 short polypeptides (short or S
chains ) joined by disulfide bonds.
➔ Composed of 82% to 96% proteins, and 4% to 18%
carbohydrates
➔ Their synthesis in the plasma cells is triggered by an
immune response to foreign bodies and organisms.
➔ It has 5 major groups based on the type of heavy chain they
have: IgG, IgA, IgM, IgD, and IgE.
➔ Heavy chains are γ, α , µ, δ, and ε respectively.
➔ There are 2 light chains: kappa (κ)and lambda (λ) in a ratio
of 2:1
➔ Each chain has 2 regions: constant and variable region.
Constant region is identical in all antibodies of the same
isotypes. The variable of the heavy chain differws in
antibodies produced by different B cells, but is the same for
all antibodies produced by a single B cell or B-cell clone.
➔ Immunoglobulins are not synthesized to any extent in
neonates.
➔ IgM is the first antibody to appear in response to antigenic
stimulation. It is an antibody that acts as anti-A or anti-B
antibody to red blood cell antigen, rheumatoid factors, and
heterophile antibodies.
➔ IgG is the most abundant class; act on bacteria, fungi,
viruses and foreign bodies through agglutination,
opsonization, and complement activation , and secretions of
respiratory and gastrointestinal mucosa; found in small
amounts in blood
➔ IgA is the main immunoglobulin found in mucous secretions,
including tears, saliva, colostrum, vaginal fluid
➔ IgD are present on the surface of most, but not all, B cells
early in their development; little amount is released into the
circulation. This helps regulate B-cell function
➔ EgE is produced by B cells and plasma cells; associated
with allergic and anaphylactic reactions. Very little in the
circulation
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➔ Igs are determined using
nephelometry, turbidimetry,
immunoassay (ECLIA), and RIA
MIDTERM: S.Y. 2022-2023
radial immunodiffusion,
Causes of Myoglobin Elevations
electrochemiluminescent
AMI Myopathies
Angina without infarction Vigorous exercise
Rhabdomyolysis Intramuscular injections
Multiple fractures, muscle trauma Open heart surgery
Renal failure Tonic-clonic seizures
Electric shock Arterial thrombosis
Certain toxins

Cardiac Troponin (cTn)

Miscellaneous Proteins
Myoglobin
➔ Is a heme protein found in striated skeletal and cardiac
muscles.
➔ It comprises approximately 2% of the total muscle proteins.
➔ It can reversely bind oxygen in a manner similar to
hemoglobin.
➔ This protein is increased in acute myocardial infarction
(AMI). The increase is seen 2 to 3 hours after the onset and
reaches the peak in 8 to 12 hours. The degree of elevation
indicates the size of the infarct.
➔ Since the size of myoglobin is small, it is filtered in the
kidneys and the level in the blood returns to normal within
18-30 hours after the attack.
➔ This makes myoglobin a useful tool for early AMI detection.
However, even if myoglobin is 75% to 100% sensitive to
AMI, it must be emphasized that myoglobin is NOT cardiac
specific.
➔ Myoglobin is measured using latex agglutination,
enzyme-linked immunosorbent (ELISA),
immunonephelometry, ECLIA and fluoroimmunoassays.
Elevations are also seen in progressive muscular dystrophy
and crushing injury in which skeletal muscle is damaged.
➔ Is a complex of 3 proteins that bind to the thin filaments of
cardiac muscle.
➔ The 3 proteins are troponin T (cTnT), troponin I (cTnI), and
troponin C (TnC).
➔ Cardiac troponins are “gold standard” for diagnosis of acute
respiratory syndrome (ACS) in which blood supply to the
heart muscle is suddenly impeded. A single blood cTn is is
indicative of myocardial injury.
➔ Following an AMI, blood cTn elevate within 3 to 12 hours,
reach peak concentration in 12 to 24 hours, and remain
elevated for 1 to 3 weeks.
➔ If CK, CK-MB and myoglobin are normal but troponin is
elevated, it is possible that damage to the heart was minor
or that injury took place more than 24 hours in the past. If
the initial troponin measurement is within reference interval
but subsequent (6 and 12 hour specimens) troponin results
are elevated, then damage to the heart likely to occur a
couple of hours prior to the initial specimen collection and
blood concentrations had not yet significantly elevated.
When CK is elevated but troponin is within reference range,
it is likely that symptoms are present due to a cause other
than AMI.
➔ cTn can be measured using ELISA , immunoenzymometric
assays
Brain Natriuretic Peptide and N-Terminal-brain Natriuretic
peptide
➔ Neurohormones that affect body fluid homoestasis, through
natriuresis and diuresis, and blood pressure, through
decreased angiotensin II and norepinephrine synthesis
➔ Marker for congestive heart failure
➔ Laboratory analysis: RIA, microparticle enzyme
immunoassay (MEIA), and ECLIA
Fibronectin)
➔ Roles in cell division, tissue differentiation, growth, wound
healing

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➔ Found in plasma and on cell surfaces
➔ Synthesized by liver cells, endothelial cells, peritoneal
macrophages, and fibrobasts
➔ Fetal fibronectin (fFN) is used to predict short-term risk of
premature delivery. fFN is normally detected in amniotic
fluid and placental tissue at early pregnancy but in normal
pregnancy, it is not detectable i after 24 weeks of gestation
➔ Elevated fFn between 22 to 36 weeks of gestation reflect a
disturbance at the uteroplacental junction and are
associated with an increased risk of preterm labor and
delivery
Adiponectin
➔ There is a universal correlation between body mass index
and adiponectin. Lower adiponectin is correlated with an
increased risk of heart disease, type 2 DM, metabolic
syndrome , and obesity
β-trace protein (BTP or prostaglandin D synthase)
➔ Established as an accurate marker of CSF leakage; marker in
detecting impaired renal function ( but not a diagnostically
efficient test for GFR)
➔ Advantage over cystatin: not influenced by glucocorticoid
therapy
Cross-linked C-telopeptides (CTXs)
➔ Presence in urine or serum is a biomarker of bone resorption
but CTXs cannot replace bone mineral density testing in the
diagnosis of osteoporosis
Cystatin
➔ Freely filtered by glomerulus and almost completely
reabsorbed and catabolized by the proximal tubular cells
➔ New sensitive serum marker for GMF because it is produced
and destroyed at a fairly constant rate
➔ Unlike creatinine, cystatin C is not affected muscle mass,
gender, age, or race and generally is not affected by most
drugs, infections, diet, or inflammation
➔ It is also associated with an increased risk of cardiovascular
disease and heart failure in the elderly
➔ Laboratory analyses: particle-enhanced
immunoturbidimetric or immunonephelometric methods
Amyloid
➔ Insoluble fibrous protein aggregates formed due to an
alteration in their secondary structure
➔ Deposited in organs and tissues
➔ Can be inherited secondary to disease that cause abundant
or abnormal protein production, such as chronic infections,
malignancies, and rheumatologic disorders
➔ Amyloid β42 (A β42)and Tau protein tests maybe used to
differentiate Alzheimer’s disease from other forms of
dementia
Total Protein abnormalities:
1. Hypoproteinemia - this means low protein level in the
blood. There is negative nitrogen balance. This happens in:
a. Protein loss in the case of excretion in urine in renal
disease, leakage into the gastrointestinal tract in
inflammation of the digestive system, and the loss
of blood in open wounds, internal bleeding, or
extensive burns
b. Decreased protein intake in case of malnutrition and
deficiency of protein absorption
c. Decreased protein synthesis as seen in liver disease
and inherited immunodeficiency disorder
d. Accelerated protein catabolism as seen in burns,
trauma or other injuries
2. Hyperproteinemia - This means high protein level in the
blood. It is not as common as hypoproteinemia. This is seen
in:
a. Dehydration
b. Excessive production of γ-globulin
c. Disorders that leak protein into the urine as in the
case of multiple myeloma
Methods of Analysis for Protein
Total Nitrogen
➔ The determination for total nitrogen measures all
chemically nitrogen in the sample. The measurement is
useful in assessing nitrogen balance.
Chemiluminescence
➔ Sample + oxygen
➔ HBeated at a very high temperature = nitrogen oxidized into
nitric oxide
➔ Nitric oxide + ozone (O3) = nitrogen dioxide (NO2)
➔ NO2 decays to the ground state = it emits photon of light
➔ Amount of light emitted = concentration of nitrogen
Total Proteins
➔ Reference interval for serum total proteins: 6.5 g/dL to 8.3
g/dL (65 to 83 g/L) for ambulatory adults
➔ Reference interval for recumbent position: 6.0 g/dL to 7.8
g/dL (60 to 78 g/L
➔ Total protein is lower at birth; reach adult level by 3 years of
age
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➔ Does not require fasting; chylous and hemolyzed samples Folin-Ciocalteau
affect some methods ➔ This protein measurement is based on the ability of
phosphotungstic acid (phenol reagent or Folin-Ciocalteau’s
Kjeldahl reagent) to oxidize the phenotic structures of the amino
➔ This is the standard reference method for total protein acids tyrosine, phenylalanine, tryptophan and histidine.
measurement because of its high precision and accuracy.
➔ In this method, the serum proteins are precipitated using Lowry Protein Assay
organic acids like TCA or tungstic acid. Then nitrogen is ➔ This method uses Biuret method followed by the
liberated using a digestion process with the use of H2SO4 Folin-Ciocalteau’s method. This method enhances the color
with heat (340-360˚C) and a catalyst (copper sulfate) to obtained. This proves that it is more sensitive than Biuret
speed the reaction. Potassium sulfate is also added to method alone.
increase the boiling point to improve the efficiency of
digestion. The nitrogen is then converted to ammonium Dye binding
bisulfite which is then measured by adding alkali and ➔ The dye-binding methods are based on the ability of most
distilling ammonia into a standard boric acid solution. The proteins in serum to bind dyes. The dyes used are:
ammonium borate formed is then titrated with HCl to bromphenol blue, Ponceau S, amido black10B, lissamine
determine the amount of nitrogen in the original protein green, and Coomassie brilliant blue.
solution.
➔ Assume average nitrogen content in this method is: 16% Ultraviolet Absorption
➔ This method is not used in the routine laboratory work ➔ Proteins absorb light at 280 nm and 210 nm. The
because it is time consuming and too tedious. absorbance at 280 nm is related to the absorbance of
tyrosine, tryptophan, and phenylalanine. The absorbance of
Refractometry proteins at 210 nm is due to the absorbance of the peptide
➔ Refractometry is used when rapid method that requires a bond in this wavelength.
very small volume of serum is needed. It is very easy and
fast. Specific Proteins Methods
➔ A refractometer or a total solid meter is used to measure
the refractive index. The refractive index reflects the
a. Methods for Albumin
concentration of protein, assuming that nonprotein solids
are present in same concentration as in the calibrating
Salt Precipitation
serum. But since other substances in the serum also
➔ To get the albumin content, globulins are precipitated with
produce refraction, a built-in scale has to be calibrated.
22-26% sodium sulfate (salting-out process). The A/G
➔ The refractive index is also temperature dependent;
(albumin: globulin) ratio is normally between 1:3 to 3:1. A
therefore, a built-in temperature correction should be
reversal in the A/G ratio is seen in liver, infectious diseases,
incorporated.
multiple myeloma, and nephritis.
➔ This is labor intensive
Biuret
➔ Among the adaptations of the Biuret reaction are the
➔ This is the most widely and routinely used method. The
Kingsley-Biuret and Weischelboum methods.
reaction is based on the ability of the peptide bonds to react
with copper ions to form a violet-colored chelate “Biuret”
Dye Binding
complex.
➔ This is the most common method, especially in research.
➔ There must be at least two peptide bonds in the
The pH of the solution is adjusted so that albumin is
polypeptide for a positive reaction to occur. The Biuret
positively charged. Then, by electrostatic forces, the
reagent contains copper sulfate (for Biuret formation),
albumin is attracted to and binds to an anionic dye. Once
sodium hydroxide (provides alkalinity but is not critical),
bound, it possesses an absorbance distinct to albumin. In
Rochelle salt (potassium sodium tartrate) to stabilize the
other words, the dye binding principle is “albumin binds to
copper sulfate which is unstable in an alkaline environment,
the dye and causes a shift in absorption maximum”
and potassium iodide (keeps the copper ions in the cupric
➔ The dyes used for albumin are methyl orange,
state)

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2-4”-hydroxyazobenzene)-benzoic acid (HABA), bromcresol
green (BCG), and Bromcresol purple (BCP). Methyl orange
is not albumin specific, HABA has many interferences
(salicylates and bilirubin, BCG is sensitive. It overestimates
low albumin levels and is most commonly used dye. BCP is
an alternate dye that specifically binds to albumin. It is
sensitive and precise and not subject to most interference.

Electrophoresis
➔ Separates protein based on the basis of their electric charge.
It is accurate because it gives overview of relative changes
in different protein fractions

b. Methods for Globulins

Direct colorimetric
➔ This method uses glyoxylic acid. With Cu there is
condensation of tryptophan found in globulin to produce a
purple color.
➔ This is not a common method for globulin measurement
because of the ease and simplicity of dye binding method

Electrophoresis
➔ Electrophoresis separates proteins on the basis of their
electric charge densities.
➔ At a pH greater than the pI, the protein is negatively
charged. The direction of the movement depends on the
charge: cations (positive charge) migrate to the cathode
(negative terminal) while the anions (negative charge)
migrate to the anode (positive terminal)

68 | MLS 111A