AMINO ACIDS & PROTEINS - excess nitrogen that is generated when protein is used

by the body.
Reference: Bishop (7th edition) & Handouts ni Sir Roi
last sem 9. Argininosuccinic Aciduria
- lack the enzyme argininosuccinic acid lyase which
Proteins- building blocks of life prevents the conversion of argininosuccinic acid into
Amino acids- building blocks of proteins arginine.

Function: TYROSINE
- growth, repair, and maintenance of all cells are
dependent on amino acids Type I tyrosinemia
- most severe
Proteins -caused by low levels of the enzyme
-catalyze by almost all of the reactions in living cells, fumarylacetoacetate hydrolase
controlling virtually all cellular processes.
- composed of carbon, oxygen, hydrogen, nitrogen, and Type II tyrosinemia
sulfur - Caused by a deficiency of the enzyme tyrosine
aminotransferase
NOTE:
-About half of the 20 amino acids needed by humans Type III tyrosinemia
cannot be synthesized at a rapid enough rate to support - a rare disorder caused by a deficiency of the enzyme 4-
growth hydroxyphenylpyruvate dioxygenase
- The other half of 20 amino acids can be use by
eating/diet. CITRULLINEMIA
- Amino acids are also released by the normal
breakdown of body proteins Type I Citrullinemia
- Primary purpose of Amino acids: Synthesis of Body - metabolic defect caused by the lack of the enzyme
proteins arginosuccinic acid synthetase

AMINO ACID DISORDER Type II Citrullinemia

- caused by mutation of the gene
1. Phenylketonuria (PKU) - prevented from making citrin, which inhibits the urea
- absence of activity of the enzyme phenylalanine cycle and disrupts the production of proteins and
hydroxylase (PAH), which catalyzes the conversion of nucleotides
phenylalanine to tyrosine
TREATMENT: high-caloric, protein-restrictive diet,
2. Tyrosinemia arginine supplementation and administration of sodium
- characterized by the excretion of tyrosine and benzoate and soducium phenylacetate
tyrosine catabolites in urine

3. Alkaptonuria ESSENTIAL AMINO ACIDS

- inborn metabolic disease transmitted as an autosomal
recessive gene (HGD gene) which causes the lack of the Arginine (Arg)
enzy me homogentisate oxidase. - found at catalytic (active) site in proteins and enzymes
- color of urine turns brownish-black when it mixes w/ - important role in cell division, healing of wounds,
air stimulation of protein synthesis, immune function, and
release of hormones
4. Maple Syrup Urine Disease (MSUD) - required for the generation of urea and synthesis of
- absence of the enzyme branched-chain-ketoacid creatinine
decarboxylase
Histidine (His)
5. Isovaleric Acidemia - one of the basic amino acids due to its imidazole side
- deficiency of the enzyme isovaleryl-CoA chain
dehydrogenase -direct precursor of histamine
- Sweaty feet odor - important source of carbon atoms
pH: <7.35 - needed to help grow and repair body tissues and to
Normal pH: 7.35-7.45 maintain the myelin sheaths
- helps to protect the body from heavy metal toxicity
6. Homocystinuria - stimulates the secretion of the digestive enzyme gastrin
- lack of the enzyme cystathionine-b-synthetase
- elevated plasma and urine levels of methionine Isoleucine (Ile)
- it helps to maintain, heal, and repair muscle tissue,
7. Cystinuria skin, and bones.
- inadequate reabsorption of cystine during the filtering - needed in hemoglobin formation and helps to regulate
process in the kidneys blood glucose levels
- Homogentisic Acid oxidase

8. Citrullinemia
- AKA as Urea Cycle Disorders
Leucine (Leu) CLASSIFICATION OF PROTEINS
- second most common amino acid
- boost the healing of muscle, skin, and bones Simple Proteins
- aids in recovery from surgery and lowers blood glucose - composed of only amino acids yields from peptide
chain hydrolysis
Lysine (Lys)
-production of antibodies and lowers TAG levels A. Globular Proteins
- needed for proper growth and bone development in - symmetrical, compactly folded polypeptide chains
children E.g. Albumin
- helps in the absorption and conservation of calcium
- important in formation of collagen B. Fibrous Proteins
- elongated, asymmetrical polypeptide chains
Methionine (Met) E.g. Troponin and Collagen
- first amino acid incorporated into the N-terminal
position of all proteins Conjugated Proteins
- source of sulfur - composed of protein (apoprotein) and non-protein
- helps to detoxify lead and other heavy metals; helps to components
diminish muscle weakness, and prevents brittle hair
A. Metalloproteins
Phenylalanine (Phe) - with a metal prosthetic group (Ceruloplasmin)
- non-polar amino acid (hydrophobic nature)
- promotes alertness and vitality, elevates mood, B. Lipoproteins
decreases pain, aids memory and learning. - with a lipid prosthetic group (Cholesterol, TAG)
- used by the brain to produce norepinephrine
- part of the compositon of aspartame C. Glycoproteins
- with 10-40% carbohydrates attached (Haptoglobin)
Threonine (Thr)
- important component in the formation of protein, D. Mucoproteins
collagen, elastin, and tooth enamel - with >40% carbohydrates attached (Mucin)
- helps to maintain proper protein balance in the body
E. Nucleoproteins
Tryptophan (Trp) - with DNA or RNA nucleic acid attached (Chromatin)
- formed from proteins during digestion
- precursor for serotonin and melatonin PROTEIN METABOLISM
- a natural relaxant
- it helps to alleviate insomnia Nitrogen balance
- used as a treatment of migraine headaches - a balance between anabolism and catabolism
- it helps to control hyperactivity in children
Negative nitrogen balance
Valine (Val) - when protein catabolism exceeds anabolism, tissue
- a constituent of fibrous protein in the body destruction such as burns, wasting disease , high fever
- needed for muscle metabolism and coordination and starvation
- used as a treatment for muscle, mental, and emotional
problems; insomnia, anxiety, liver and gall bladder Positive nitrogen balance
disease - protein anabolism exceeds catabolism

PROTEIN STRUCTURES SIGNIFICANT PROTEINS

Primary structure Pre-albumin (Transthyretin)

- the amino acids are linked to each other through - indicator of nutritional status and transports thyroid
covalent peptide bonding in a specific sequence to form hormones
a polypeptide chain - concentrated protein in CSF than in SERUM

Secondary structure Albumin

- the polypeptide chain winds to form alpha helixes and - highest concentration of all plasma proteins
beta sheets through the formation of hydrogen bonds
between CO and NH groups of the peptide bonds α1-Globulins: α1-Antityrpsin
- acute phase reactant and protease inhibitor
Tertiary structure - decreased in emphysema
- the coiled polypeptide chain folds upon itself to form a - associated with severe Juvenile Hepatic disorders that
3D structure through the interaction of R groups of the may result in Cirrhosis
amino acids
α1-Globulins: α1-Fetoprotein
Quaternary structure - synthesized during gestation in the yolk sac and liver of
- 2 or more folded polypeptide chains bind to each other the fetus
through hydrogen bonds and electrostatic interactions to - Fetoprotein is the marker for Down syndrome
form a functional CHON
α1-Globulins: α1-Acid Glycoprotein (Orosomucoid)
- acute-phase reactant that binds to basic drugs
 α1-Globulins: α1-Antichymotrypsin Dye-binding Method
- binds and inactivates PSA - ability of protein to bind dyes
- marker for Alzheimer’s Bromcresol Green: most widely used
Bromcresol Purple: more specific in binding albumin
α1-Globulins: α1-Gc-globulin
(Group-Specific Component, Vitamin D-Binding Serum Protein Electrophoresis
Protein) - support medium (Cellulose acetate or Agarose Gel)
- affinity w/ Vit. D and actin
Gamma-spike Monoclonal Gammopathy
α1-Globulins: α1-Ceruloplasmin (Multiple Myeloma)
- transport protein of copper Beta-gamma Bridging In serum: Hepatic
- decreased in Wilson Disease and Severe Liver Disease Cirrhosis (IgA)
In plasma: Normal
α1-Globulins: α1-Haptoglobin (Fibrinogen)
- marker for hemolysis Alpha2-globulin bind spike Nephrotic Syndrome
- binds free Hgb and acute-phase reactant Alpha1-globulin Flat Curve Juvenile Cirrhosis
α1, α2, Beta-globulin
α1-Globulins: α1-Macroglobulin band spikes Inflammation
- proteolytic enzyme inhibitor that inhibits thrombin, Polyclonal Gammopathy Chronic Inflammation
trypsin, and pepsin Small spikes in beta region Transferrin
- increased in Nephrotic Syndrome

β-Globulins: β2-Microglobulin
- filtered by glomeruli but reabsorbed

β-Globulins: Hemopexin
- binds free heme

β-Globulins: Complement
- immune response; C3 most predominant complement

β-Globulins: Fibrinogen
- largest proteins in blood plasma
- clotting factor

β-Globulins: Transferrin/Siderophilin
- β-Globulins that transports iron

β-Globulins: C-reactive Protein

- globulin that is an acute-phase reactant

γ-Globulins: Immunoglobulins
- synthesized in the plasma cell
- IgG >IgA >IgM >IgD > IgE

IgG- can cross the placenta

IgA- levels increase after birth
IgM- largest antibody in terms of size
IgD- increased in liver disorders, infections, and
connective tissue disorders
IgE- increased in allergies, asthma, and hay fever, and
during parasitic infections

METHODOLOGY

Kjeldahl Method
- reference method of choice
- quantification of the nitrogen content of protein
1gN2= 6.54g of protein
15.1-16.8%= N2 content of proteins
H2SO4= reagent
NH3= end product

Biuret Method
- most widely used
- cupric ions complexing w/ peptide bonds in an alkaline
medium to produce a purple-colored complex
Rgts: Alkaline CuSO4, NaOH, NaK Tartrate, KI
End product: Violet color (545nm)
CARBOHYDRATES - All monosaccharides and disaccharides are
Reference: Bishop (6th ed.) reducing substances except sucrose
Introduction: Glucose metabolism:
- Organisms rely on oxidation for them to obtain Embden-Myherhof Pathway
energy
- Carbohydrates, Amino acids, Lipids are sources
of energy but brain, erythrocytes, and retinal cells
mainly depend on carbohydrates
- It is major food source and energy supply of the
body and stored in liver and muscles
- Hyperglycemia and hypoglycemia are known
diseases involving carbohydrates
- Compounds containing C, H, O
- Can be formed with phosphate, sulfate, amines
- Hydrates of aldehyde (aldose) and ketone
(ketose)
Properties:
o Size of base carbon chain
o Location of the CO function group
o Number of sugar units
o Stereochemistry of the compound
Classification:
o Trioses: three carbons - The first step in glycolysis is the phosphorylation
o Tetroses: four carbons of glucose by ATP to form glucose 6-phosphate.
o Pentoses: five carbons This reaction, catalyzed by the enzyme
o Hexoses: six carbons hexokinase, traps glucose in the cell.
o Glyceraldehyde: three-carbon compound - The second step in glycolysis is the isomerization
(smallest carbohydrate) of glucose 6-phosphate to fructose 6-phosphate.
This converts the sugar from a 6-membered
pyranose to the 5-membered furanose structure
and involves the conversion of an aldose into a
ketose. This reaction is catalyzed by the enzyme
phosphoglucose isomerase.
- The third step in glycolysis is a second
phosphorylation to form fructose 1,6-
bisphosphate catalyzed by the enzyme
phosphofructokinase. Phosphofructokinase is an
allosteric enzyme controlled by ATP and other
metabolites. The importance of the control of
phosphofructokinase will be discussed in a later
Stereoisomers: lesson.
- Central carbons are asymmetric (chiral)
- Same order and types of bonds but different
spatial arrangement and diff. properties Regulation of Carbohydrate Metabolism:
- Can’t be overlapped and non-superimposable - Liver, Pancreas, and Endocrine Glands are
Monosaccharides – simple sugars; 3-6 carbons; glucose, involved in controlling the blood glucose
fructose, galactose - Insulin are hormone that manages to decrease the
levels of carbohydrate while glucagon opposes
Disaccharides – 6 or more carbons; maltose, lactose, opposite function since it increases the glucose
sucrose levels, both of produces by pancreas
- Insulin:
Polysaccharides – 2-10 carbons; starch and glycogen
 is the primary responsible for entry of
glucose, it acts as a gate
 Synthesized by Islet of Langherhans
Chemical Properties:  Increase movement of glucose in cells and
metabolism
- Carbohydrate reducing substances if they contain
 Hypoglycemic agent
ketone and aldehyde
- Glucagon:
- Carbohydrate non-reducing substances if they do
 Synthesized by B-cells of islet of
not contain active ketone or aldehyde
Langherhans
- It can form glycosidic bonds with
 Released during stress and fasting situation
noncarbohydrates compound
 Hyperglycemic agent
- Reducing substances: glucose, maltose, fructose,
lactose, galactose
 - Glucocortisoids/Cortisol - Abrupt onset
 Produced by adrenal cortex - Signs and symptoms: Polydipsia, Polyuria,
 Hyperglycemic agent Polyphagia, rapid weight loss, hyperventilation,
- Epinephrine mental confusion, loss of consciousness
 Produced by adrenal medulla
 Hyperglycemic agent Type 2-DM
 Released during stress - Hyperglycemia by insulin resistance
- Growth Hormone - Impaired-fasting glucose
 Produced by pituitary gland - Insulin resistance
 Hypoglycemic agent
- Inherited, increases risk in older age, obese, lacks
- ACTH physical exercise
 Hyperglycemic agent - 90% to 80% cases
- Thyroxine
 Hyperglycemic agent Gestational Diabetes
- Somatostatin
 Synthesized by B-cells of islet of - Glucose intolerance during pregnancy
Langherhans - Metabolic and hormonal changes
 Hyperglycemic agent - Normal if postpartum
- Respiratory distress, hypocalcemia,
hyperbilirubinemia
Pathophysiology of Diabetes Mellitus:
- Type 1 and 2 DM will result to hyperglycemia,
glucosuria after renal tubular system becomes
saturated
- Type 1 DM produces high level of ketones while
Hyperglycemia: Type 2 DM generates seldom number of ketones
but higher risk to develop hyperosmolar
- Increase plasma glucose caused by imbalance nonketotic states
hormones - Type 1 DM has absence insulin with excess
glucagon
Diabetes Mellitus
- Type 2 DM has insulin but attenuated glucagon
- Metabolic disorder by hyperglycemia - Impaired glucose metabolism includes impaired
- Defect in insulin secretion fasting and glucose tolerance
- Divided into: The demonstration of hyperglycemia or hypoglycemia is
 Type 1 - Insulin-dependent-DM
used to diagnosed diabetes mellitus and hypoglycemic
 Type 1 - Non-insulin-dependent-DM
conditions.
 Other specific types of diabetes
 Gestational DM
HYPOGLYCEMIA
- occurs because glycogen cannot be converted back to
glucose by hepatic glycogenolysis
- involves decreased plasma glucose levels (70mg/dl)
- some causes may be transient and others can be life
threatening
GALACTOSEMIA
- is cause by a failure to thrive syndrome in infants
- congenital deficiency of one of three enzymes
- occurs because of uridyltransferase
GLUCOSE
- can be measured from serum, plasma, or whole blood if
serum or plasma is the specimen being used it must be
refrigerated and seperate from the cells within 1 hour
- fasting blood glucose (FBG) should be obtained in the
morning after an approximately 8 to 10 hours fast
- the ability of glucose to function as reducing agent;
most common methods of glucose analysis use the
Type 1-DM enzyme glucose oxidase (most specific enzyme) or
- Inappropriate increase by destruction of islet B- hexokinase (more accurate than glucose oxidase)
cell
GLYCOSYLATED HEMOGLOBIN/HbA1C
- Ketoacidosis
- is the term used to describe the formation of
- Insulin deficiency
hemoglobin compound produced when glucose (a
- 10% to 20% cases
reducing sugar) reacts with the amino group of
- Environmental factor by virus
hemoglobin (a protein)
- the glycosylated hemoglobin level at any one time Cholesterol
reflects the average blood glucose level over 2 or 3 - esterified form: contain 1 fatty acids
months
- most commonly detected glycosylated hemoglobin, is a FUNCTIONS:
glucose molecule attached. It is also the most reliable > repair cell membrane
method to monitor the long-term diabetes control > synthesis of bile acid and Vit. D
> precursor of 5 classes of steroid hormones
- the specimen requirement is an EDTA whole blood
sample
5 Classes of Steroid Hormones
 Methods of measurements: Progestins
Androgen
 1. based on charge differences between
Glucocorticoids
glycosylated and non glycosylated hemoglobin Estrogen
 2. structural characteristics of glycogroups om Mineralocorticoids
hemoglobin
 Reference Method: Affinity Chromatography 2 FORM

KETONES Free Cholesterol

- are produced by the liver through metabolism of fatty - 1/3 or 30%
acids to provide a ready energy source from stored lipds
at times of low carbohydrate Cholesterol Ester
- 2/3 or 70%
- the measurements of ketones is recommended for
patients with Type 1 DM during acute illness, stress,
LIPOPROTEINS
pregnancy, or elevated, blood glucose levels above 300 - composed of both lipids and proteins called
mg/dL APOLIPOPROTEINS
- the specimen requires fresh serum or urine; the sample
should be tightly stoppered and analyzed immediately CHYLOMICRONS
- contain ApoB-48; largest and least dense of the
lipoprotein particles
 3 Ketone Bodies: - produce in intestine
 1. Acetone (2%)
Principal role: deliver lipids
 2. Acetoacetic acid (20%) Characteristic: low density, chylomicrons will float to
 3. 3-B-hydroxybutyric acid (78%) the top and form a creamy layer

KETONEMIA VLDL
- refers to the accumulation of ketones in blood, and the - produce by the liver
term ketonuria refers to the accumalation of ketones in - contains ApoB-100, Apo E, and Apo Cs
urin - second least dense

LIPIDS LDL (Bad Cholesterol)

- substances that are insoluble in water - contain ApoB-100
- forward cholesterol transport
IMPORTANT ROLE: - cholesterol rich
> maintain cell membrane integrity - marker for coronary heart disease
> precursor of steroids hormone
> sources of energy HDL (Good Cholesterol)
- smallest and most dense lipoprotein particles
- reverse cholesterol transport
LIPID STRUCTURE - anti-atherogenic effect

Fatty Acids OTHER LIPOPROTEINS

- major constituents of TAG and phospholipids
- substrate for Gluconeogenesis Intermediate-Density Lipoproteins
- sources of metabolic energy - carry endogenous TAG and cholesterol esters
- have ApoB-100 mainly, some E on their surface
Saturated
- without double bonds Lp(a)/Lipoprotein A
- AKA as sinking pre-beta lipoprotein
Unsaturated - independent risk factor for atherosclerosis
- with double bonds
Beta-VLDL
NOTE: - AKA as floating beta lipoprotein
The presence of double bonds makes the unsaturated - found in type 3 hyperlipoproteinemia (ApoE-IIIdef;
fatty acid more liquid at room temperature IDL)
- faulty VLDL metabolism
 Salkowski reaction: Cholestadienyl Disulfonic Acid
LpX (+): Red end color @410nm
- found in obstructive jaundice and LCAT deficiency
(LCAT- Lecithin Cholesterol Acyl Transferase) One step
- Pearson, Stern, Mac Gavack
ADULT REFERENCE RANGES FOR LIPIDS
Two step
ANALYTE - Colorimetric, Extraction
Total Cholesterol 140-200mg/dL - Bloor’s method
HDL 40-75mg/dL
LDL 50-130mg/dL Three step
Triglycerides 60-150mg/dL - Colorimetric, Extraction, Saponification
- Abell Kendall
NOTE:
Cholesterol and TAG requires 8-12 hours fasting Four step
- Colorimetric, Extraction, Saponification, Precipitation
CLINICAL SIGNIFICANCE - Sperry, Parekh & Jung

Atherosclerosis ENZYMATIC METHOD

- disease which there is an accumulation of lipid material
in the veins and arteries > Cholesterol esterase
> Cholesterol oxidase
Congestive Heart Disease > Peroxidase
- inability of heart to pump blood effectively
Abell, Levy and Brodie Method
Hypercholesterolemia - Reference method
- lipid abnormality and most closely linked to heart
disease TRIGLYCERIDES MEASUREMENT

Hyperlipoproteinemia CHEMICAL METHOD

- increase lipoprotein because of improper synthesis or
breakdown of lipoprotein fraction Van Handel & Zilversmith
- increased TAG and Cholesterol - colorimetric
- Chromotropic Acid
Abetalipoproteinemia (+): Blue color compound
- cholesterol is very low
- TAG is undetectable Hantzch Condensation
- LDL by ApoB-100 is absent - fluorometric
- Diacetyl acetone
Hypobetalipoproteinemia (+): Diacetyl lutidine compound/Yellow Color
- unable to synthesize ApoB-100 and ApoB-48
- total cholesterol is low
- TAG is normal to low ENZYMATIC METHOD
Hypoalphalipoproteinemia > Bacterial Lipase
- TAG is severe elevated > Glycerokinase
- HDL is low > Glycerophosphate oxidase
> Peroxidase
Tangier’s Disease
- HDL is absent Modified Van Handel and Zilversmith
- ApoA-1 and ApoA-II is very low - reference method
- Total cholesterol and LDL is low (+): Pink color
- TAG is normal to slightly increase

METHODS OF ANALYSIS

Lipid Panel

Directyl Measures Indirectly measures

Total cholesterol LDL Cholesterol
HDL Cholesterol VLDL
Triglycerides

TOTAL CHOLESTEROL MEASUREMENT

Chemical rxn:
Liebermann Burchardt: Cholestadienyl Monosulfonic
Acid
(+): Green end color @410nm