LECTURE NOTES 2: CLINICAL CHEMISTRY ● Glucose-6-phosphate is then shunted into metabolic

OVERVIEW: pathways:
● CARBOHYDRATES PATHWAYS DESCRIPTION
● LIPIDS & LIPOPROTEINS Metabolism of glucose molecule
● NON-PROTEIN NITROGEN GLYCOLYSIS to pyruvate or lactate for
● CLINICAL ENZYMOLOGY production of energy
● ELECTROLYTES Formation of glucose-6-phosphate
GLUCONEOGENESIS
● LIVER FUNCTION from non-carbohydrate source
● KIDNEY FUNCTION Breakdown of glycogen to glucose
GLYCOGENOLYSIS
● ENDOCRINOLOGY for use as energy
Conversion of glucose to glycogen
GLYCOGENESIS
CARBOHYDRATES for storage
● Contains Carbon, Hydrogen, and Oxygen Conversion of carbohydrates to
LIPOGENESIS
● Major source of stored and used energy fatty acids
● Component of cell membranes LIPOLYSIS Decomposition of fat
● Structural component in plants, bacteria, insects
● Can be reducing or non-reducing sugars HORMONES INVOLVED IN CARBOHYDRATE
● Classified according to the number of sugar units: METABOLISM
1. INSULIN
SUGAR SUGAR EXAMPLES ◆ produced by beta cells islets of Langerhans
UNIT ◆ targets most cells of the body
Glucose, Fructose, ◆ increases utilization of glucose by cells by
Monosaccharides 1 unit
Galactose increasing cellular uptake and hepatic glycolysis
Glucose + 2. GLUCAGON
Sucrose
Fructose ◆ produced by alpha cells of the islets of
Glucose + Langerhans
Disaccharides 2 units Lactose
Galactose ◆ targets the liver
Glucose + 3. CORTISOL
Maltose
Glucose ◆ glucocorticoids
3 to 10 ◆ increases glucogeneogenesis
Oligosaccharides
units ◆ decreases utilization of glucose by cells by
more Starch, increasing cellular uptake and extrahepatic
Polysaccharides than 10 Glycogen, tissues
units Cellulose ◆ from adrenal cortex
4. CATECHOLAMINES
CARBOHYDRATE METABOLISM ◆ stimulates glycogenolysis
● Glucose is the only carbohydrate to be used directly ◆ from adrenal medulla
for energy 5. THYROID HORMONE
● In an average diet, about 50% of carbohydrates are ◆ inhibit glucose absorption in the small intestine
constituted by calories ◆ from thyroid gland
● Disaccharides are hydrolyzed into monosaccharides 6. SOMATOSTATIN
then absorbed by the gut via active transport or ◆ inhibit glucagon and insulin secretion
facilitated diffusion. They are transported to the liver ◆ from delta cells of pancreas
through portal circulation
● After glucose enters cell, it undergoes 7. GROWTH HORMONE
phosphorylation into glucose-6-phosphate through ◆ increases liver gluconeogenesis
the action of hexokinase and glucokinase. ◆ inhibits glycolysis
 ◆ inhibits glucose transport FASTING 2-HOUR
PLASMA PLASMA
DIAGNOSIS HbA1c
DIABETES MELLITUS GLUCOS GLUCOS
● heterogeneous group of multifactoral, polygenic E E
syndromes characterized by an elevated fasting <140
NORMAL <100 mg/dl
blood glucose caused by a relative or absolute mg/dl
deficiency in insulin 5.7 -
PRE-DIABETES
6.4%
DIABETES TYPE 1 DM TYPE 2 100-125
IMPAIRED FASTING
MELLITUS mg/dl
GLUCOSE
Absolute
deficiency of IMPAIRED GLUCOSE 140-199
insulin caused Combination of TOLERANCE mg/dl
by an insulin resistance DIABETES >200
DESCRIPTION >126 mg/dl >6.5%
autoimmune and dysfunctional MELLITUS mg/dl
attack on the beta cells
beta cells of the ORAL GLUCOSE TOLERANCE TEST
pancreas ● Individuals should ingest atleast 150 g/day of CHO for
● Juvenile 3 days before OGTT
● Adult Onset DM
Onset DM ● Fasting: 8-14 hrs
OTHER NAME ● Non-Insulin-
● Insulin- ● venous sample are preferred - collected in gray top
Dependent
Dependent tube containing fluoride and anticoagulant
FREQUENCY 5 to 10% 90-95% ● FBG is measured before administration of glucose
Most common load
Most common with
AGE OF ONSET in children and ■ Adults: 75 g
advancing age
young adults ■ Children: 1.75/kg body weight max of 75 g
● Genetic ● Genetic ■ Pregnant Women: 75 g ((100g)
● Autoimmune ● Obesity ● Glucose load should be finished within 5-15 minutes
RISK FACTORS
● Environment ● Sedentary ● If patient vomits, discontinue
al Lifestyle
Insulin GLYCOSYLATED HEMOGLOBIN
MEDICATION absolutely Oral agents ● rate of formation of HbA1C is proportional to the
necessary average blood glucose concentration over the
Lifestyle, oral previous 3 months
THERAPHY None known
medicines ● 1% increase HbA1c = 35 mg/dl change in plasma
glucose
GESTATIONAL DIABETES MELLITUS ● tested twice a year = long term glycemic control
● glucose intolerance with onset or first recognition ● collected in EDTA - whole blood spin = hemolysate
during pregnancy
● large percentage of patients develop DM within 5 to HYPOGLYCEMIA
10 years ● about 50-55 mg/dl glucose are released
● infants born to mothers with diabetes are at risk for ● most causes are secondary to other illnesses; resolve
severe complications when primary sickness is treated
● Screening: 2-hour OGTT using a 75 g glucose load. ● warning signs and symptoms are all related to the
CNS
● 5-h OGTT = glucose values strikingly drop around
hours 4 & 5
SPECIMEN CONSIDERATION AND PATIENT FATTY ACIDS
PREPARATION FOR GLUCOSE TESTING ● building blocks o lipids
● Possible specimens = Whole blood, serum, plasma, ● hydrocarbon chains with a terminal COO-
urine CSF, serous fluid, synovial fluid
● Standard clinical specimen = fasting venous plasma TRIGLYCERIDES
sample ● 3 fatty acid molecules attached to one molecule of
● Fasting hours: 8 -10 hours glycerol by ester bonds
● Collection: Gray Top ● serves as main storage form of energy, shock
● Whole Blood Glucose Levels: 10-15% lower than absorber, insulator and integral part of cell
fasting plasma glucose membrane
● Glucose Metabolism: Room Temp - 7 mg/dL/hr; 4C - 2
mg/dL/hr PHOSPHOLIPIDS
● Glucose CSF levels: 60-70% of plasma glucose ● contains non - polar and polar end
● constituent of cell membrane
METHODS FOR GLUCOSE MEASUREMENT
CHOLESTEROL
CHEMICAL METHODS ● serves as part of cell membranes and as parent chain
● Folin- Wu: phosphomolybdate + for cholesterol-based hormones
reduced copper = ● exist in two forms:
OXIDATION- phosphomolybdenum blue ■ Cholesterol esters: approximately 70% of total
REDUCTION ● Nelson - Somogyi: arsenomolybdate + cholesterol
reduced copper = arsenomolybdenum ■ Free Cholesterol: approximately 30% of total
blue cholesterol
ENZYMATIC METHODS
● Colorimetric Method: uses a side LIPOPROTEIN STRUCTURE

GLUCOSE reaction that consumes H2O ● typically spherical in shape

OXIDASE ● Polarographic Method: measure the ● sizes ranging from 10 to 1200 nm
METHOD rate of disappearance of oxygen using ● size of lipoprotein particle correlates with its lipid
an oxygen electrode content
● most specific method ● various lipoprotein were originally separated
● more accurate that glucose oxidase through ultracentrifugation
HEXOKINASE ● coupling reaction with G6PDH = less
METHOD interference TYPES OF LIPOPROTEINS
● generally accepted as reference ● largest
method ● least dense
CHYLOMICRONS ● highest triglyceride content
LIPID CHEMISTRY ● highest lipid content
● Lipids are a broad group of organic compounds ● causes post-prandial turbidity
which include fats, waxes, sterols, fat-soluble ● 2nd largest
vitamins, monoglycerides, diglycerides, VERY LOW ● least dense
phospholipids, and others. DENSITY ● highest trigylceride content
● The functions of lipids include storing energy, LIPOPROTEINS ● causes fasting hyperlipedmic
signaling, and acting as structural components of cell turbidity (pathologic)
membranes. ● smallest
LOW DENSITY ● highest cholesterol content
LIPOPROTEINS ● associated with risk for
artherosclerosis
 ● smallest but densest NCEP GUIDLINES
HIGH DENSITY ● higher protein content REFERENCE
LDL TC HDL TG
LIPOPROTEIN ● lower risk for artherosclerosis RANGE
● high fiber diet = increase LOW <40
ABNORMAL LIPOPROTEIN OPTIMAL /
<100 <200 <150
Beta-VLDL ● familial dysbetalipopotenemia DESIRABLE
Lp(a) ● LDL-like particle NEAR OPTIMAL 100-129
● biliary cirrhosis BORDERLINE 200-
130-159 150-199
● cholestasis HIGH 239
LpX ● mutations in enyzme lecithin: HIGH 160-189 >240 >60 200-499
cholesterol acyltransferase VERY HIGH >190 >500
(LCAT)
FREDERICKSON CLASIFICATION OF LIPID DISORDERS
APOLIPOPROTEIN STRUCTURE ◆ Hyperchylomicronemia
● located on the surface of lipoprotein particles TYPE 1 ◆ caused by Familial LPL deficiency
● maintain structural integrity of lipoproteins ◆ low cardiac risk
● serve as ligands for cell receptors ◆ Familial Hypercholesteronemia
TYPE 2
◆ high cardiac risk
MAJOR HUMAN LIPOPROTEIN ◆ Familial Combined Hyperlipedemia
FUNCTION TYPE 3
APOLIPOPROTEINS LOCATION ◆ high cardiac risk
Apo A-I HDL LCAT activator ◆ Familial Hypertriglyceridemia
TYPE 4
LDL receptor ◆ low cardiac risk
Apo B-100 LDL,VLDL
ligand ◆ Endogenous Hypertriglyceridemia
TYPE 5
Remnant receptor ◆ low cardiac risk
Apo B-48 Chylos
ligand TYPES TG TC LDL VLDL CM
LDL receptor TYPE 1 ^ N N N ^
Apo E VLDL, HDL
ligand TYPE 2A N ^ ^ N N
Plasminogen TYPE 2B ^ ^ ^ ^ N
Apo(a) Lp(a)
inhibitor TYPE 3 ^ ^ N ^ N
TYPE 4 ^ N N ^ N
LIPOPROTEIN METABOLISM TYPE 5 ^ ^ N ^ ^
1. ABSORPTION PATHWAY
◆ dietary lipids are absorb in the intestines through SPECIMEN CONSIDERATION AND PATIENT
lipases, enzymes, bisalts PREPARATION FOR GLUCOSE TESTING
◆ stool is expelled after ● Preferred samples: serum or plasma(EDTA)
2. EXOGENOUS PATHWAY ■ plasma for electrophoresis and
◆ chylomicrons transports exogenous or dietary fibers ultracentrifugation
to the liver ● Capillary blood samples: generally lower values
3. ENDOGENOUS PATHWAY ● Lipemic samples: seen when triglyceride levels
◆ VLDL transports endogenous or hepatic triglycerides exceed 4.6 mmol/L (400 mg/dL)
from liver and converts through LPL into LDL ● Hemolyzed and Icteric samples: unaccepable
4. REVERSE CHOLESTEROL TRANSPORT PATHWAY ● Fasting: 12 hours before venipuncture
◆ LDL transports cholesterol and will be absorbed by ● Non-Fasting: for TC and HDL-C
cell and then converted into HDL through LCAT ● Prolonged torniquet = hemoconcentration
● Reclined patients = decreased values
● Avoid water contamination
METHODS FOR LIPID CONTENT MEASUREMENT PROTEINS
● linear polymers of amino acids
CHOLESTEROL MEASUREMENT ● perform diverse functions such as regulation of
● initial extraction with zeolite to metabolism, facilitate contraction of muscles, provide
remove sterols structural framework, shuttle molecules in the blood
ABELL-
● redissolve cholesterol stream, component of the immune system.
KENDAL
● hydrolysis of cholesterol esters to
cholesterol PROTEIN STUCTURE DESCRIPTION
LIEBERMANN- ◆ number and types of amino
● (+) formation of product which
BURCHARD PRIMARY acids in the specific amino
strongly absorbs at 410 nm
REAGENT acid sequence
● reference method ◆ regularly repeating structures
GC-MS ● specifically measures cholesterol and stabilized by hydrogen bonds
SECONDARY
does no detect related sterols between the amino acids
TRIGLYCERIDE MEASUREMENT within the protein
● Colorimetric Method ◆ overall shape/conformation of
TERTIARY
■ Van Handel & Zilversmit protein molecule
■ reagent: chromotropic acid = blue ◆ shape or structure that results
colored compound from the interaction of more
CHEMICAL
● Fluorometic Method than one protein molecule or
METHODS QUARTERNARY
■ Hantzch protein sub units held
■ reagent: acetylacetone (strong together by non covalent
absorption maximum at 412 nm bonds
and also has good fluorescence
● reference method SYNTHESIS OF SERUM PROTEINS
● hydrolysis of fatty acids on ● most plasma proteins are synthesized in the liver and
GC-MS
triglycerides and measurement of secreted by the hepatocyte into the circulations
glycerol ● nitrogen content of serum proteins is on average 16%
LIPOPROTEIN METHODS ● Ribosomes: site of protein synthesis within the cell
● range in density observed among
ULTRACENTRIF
lipoprotein classes DIAGNOSTICALLY SIGNIFICANT SERUM PROTEINS
UGATION
● enables fractionation PROTEIN DESCRIPTION
ELECTROPHOR Pre-albumin indicator of malnutrition
● difference in sizes and charge
ESIS Albumin major contributor to oncotic
● depends on particle size, charge, pressure
CHEMICAL
differences in apolipoprotein content Alpha-1-antitrypsin protease inhibitor
PRECIPITATION
● used in research labs only Alpha-1-fetoprotein principal fetal protein
● uses antibodies specific to Alpha-1-acid- related to immune response
IMMUNOASSAY apolipoprotein to bind and separate glycoprotein
lipoprotein classes Haptoglobin binds hemoglobin
CHROMATO ● difference in molecular sieving Ceruloplasmin transports copper, peroxidase
GRAPHIC methods or composition in affinity activity
METHODS methods Alpha-2-macroglobulin inhibits thrombin, trypsin,
pepsin
FRIDEWALD CALCULATION Transferrin transports iron
Hemopexin binds heme
Complement immune response
Fibrinogen precursor of fibrin Uric Acid 10%
C-reactive protein opsonin Creatinine 5%
Creatine 1-2%
METHODS OF PROTEIN ANALYSIS Ammonia 0.2%

TOTAL PROTEIN ANALYSIS UREA

● digestion of protein ● present in the highest concentration in blood
KJELDAHL ● measurement of nitrogen contetnt ● major excretory of protein metabolism
● reference method ● carried in the blood to the kidney and filtered from
● formatio of violet-colored chelate the plasma by the glomerulus
between Copper ions and peptide ● most are excreted in urine
BIUERT
bonds ● used for renal function evaluation and assessment of
● routine method hydration status
ALBUMIN MEASUREMENT ● determined frequently in clinical labs through
● sensitive enzymatic methods
BROMCRESOL
● overestimates low albumin levels
GREEN (BCG)
● most commonly used dye URIC ACID
● specific ● product of catabolism of purine nucleic acids
BROMCRESOL
● sensitive (guanine and adenosine)
PURPLE (BCP)
● precise ● filtered by glomerulus and secreted by the distal
tubules into urine, but mostly reabsorbed in the
proximal tubules and reused
PROTEIN ELECTROPHORESIS
● mostly present as monosodium urate in plasma =
● sample used: serum; pH: 8.6
insolubule at around pH 7
● proteins are negatively charged = leads toward anode
● performed when an abnormality in the total protein or
CREATININE/CREATINE
albumin is found
● marker for kidney function
● PRINCIPLE: separation of proteins based on their
● formed from creatine and creatine phosphate in
charge density
muscle
● STAINS: Coomassie Blue, Amido Black, Ponceau S
● excreted in plasma at a constant rate related to
ELECTROPHORETIC PATTERNS:
muscle mass
Beta-gamma bridging liver cirrhosis
● daily secretion is fairly stable = assess renal filtration
Monoclonal spike multiple myeloma function
increase a2- ● Normal BUN/CREATININE RATIO = 10:1 to 20:1
macroglobulin, beta- ● Jaffe-Reaction: non-specific determination method
nephrotic syndorm
lipoprotein; decrease
albumin AMMONIA
decrease a1 ● formed through deamination of amino acids during
emphysema
antitrypsin protein metabolism
increase beta- ● removed from circulation and converted into urea in
use of plasma instead of serum
lipoprotein the liver
● free ammonia = toxic
NON-PROTEIN NITROGEN
● Isotope dilution mass spectrophotometry - proposed
reference method CLINICAL ENZYMOLOGY
COMPOUND PLASMA CONCENTRATION ● Enzymes are protein catalysts
Urea 45-50% ● Majority are proteins = denatured by certain agents
Amino Acids 25%
● Highly specific for their substrates and products ■ Cholinesterase
■ Chymotrypsin
GENERAL DESCRIPTION ■ Elastase-1
PROPERTIES ■ 5-nucleotidase
Active site substrate interacts withe enzymes ■ Triacylglycerol lipase
Allosteric site site other than active site ■ Trypsin
Isoenzyme multiple forms of enzyme with Lyases ■ Aldolase
different genetic origin Isomerase ■ Triosephosphate isomerase
Isoform enyzme is subject to post-transitional Ligases Glutathione synthetase
modification
Cofactors nonprotein molecule that must bind to ENZYMES WITH CLINICAL SIGNIFICANCE
particular enzymes for enzyme reaction ACID PHOSPHATASE (ACP)
to occur ● richest source in prostate
Holoenzyme apoenzyme + prostethic group ● forensic clinical chemistry = rape investigation
Proenzyme inactive form ● increased in prostatic carcinoma, bone disease

The International Union of Biochemistry (IUB) Enzyme ALKALINE PHOSPHATASE (ALP)

Commission categorized all enzymes into six (6) classes ● highest concentration in intestines, liver, bone,
based on the catalytic activity of an enzyme: spleen, placenta, and kidney
● requires magnesium in collection
CLASSIFICATION DESCRIPTION
● increase in hepatobilliary disease, bone disorders =
Oxidoreductase oxidation-reduction reaction
Paget’s disease (Ostetitis deformans)
Transferases transfer of a group other than
● physiologic elevation = pregnant women and
hydrogen
growing children
Hydrolases hydrolysis of various bonds
Lyases removal of groups from substrate with GAMMA-GLUTAMYLTRANSFERASE (GGT)
hydrolysis ● primarily in kidney, brain, prostate, pancreas, liver
Isomerases interconversion of geometric, optical, ● useful marker for liver damage
or positional isomers
● detection of alcoholism & monitoring of alcohol
Ligases joining of two substrate molecules consumption during treatment
● increase with biliary tract obstruction, chronic
alcholism
CLASSIFICATION DESCRIPTION
Oxidoreductase ■ Lactate dehydrogenase ALANINE AMINOTRANSFERASE (ALT)
■ Glucose-6-phosphatase ● liver-specific enzyme
dehydrogenase ● increase in liver disease
■ Glutamate dehydrogenase ● formerly SGPT
Transferases ■ Aspartate aminotransferase
■ Alanine aminotransferase ASPARTATE AMINOTRANSFERASE (AST)
■ Creatinine kinase ● highest concentration: cardica tissue, liver, skeletal
■ Gamma-glutamyltransferase muscle
■ Glutathione-S-transferase ● increase in liver disease, myocardial infarction,
■ Glycogen phosphorylase muscular dystrophy
■ Pyruvate kinase ● formerly SGOT
Hydrolases ■ Alkaline phosphatase
■ Acid phosphatase LACTATE DEHYDROGENASE
■ Amylase ● highest in liver, heart, skelatal muscles, RBC
● highest level = Pernicious anemia
● elevated longest = myocardial infarction
■ rises within 6-8 hrs
■ peaks 24 hours
■ returns normal 5 day

CREATININE KINASE
● cardiac muscle, skeletal muscle, brain
● sensitive indicator: Myocardial Infarction
● highest elevation: Duchenne Muscular Dystrophy
● most sensitive enzyme for skeletal muscle disease
● ISOENZYMES:
■ CK-BB
◆ most anodal
◆ highest concentration: CNS
■ CK-MB
◆ value detection in AMI
◆ rise 4-8 hrs, peak 12-24 hrs, normal 48-72
hrs
■ CK-MM
◆ major form in sera of healthy people
◆ hypothyroidism

AMYLASE
● salivary glands, pancreas
● breaks down starch to simple sugar
● increase in acute pancreatitis

LIPASE
● specific for pancrease
● increase in acute pancreatitis
● olive oil substrate is used in determination;
■ modification: triolein

GLUCOSE-6-PHOSPHATE DEHYDROGENASE
● first step of glucose metabolism
● hemolysate = whole blood = measurement
● G6PD Deficiency, drug-induced hemolytic anemia

PSEUDOCHOLINESTERASE
● diagnosis and management of organophosphate
poisoning