CLINICAL CHEMISTRY

Table of Contents
PART I: BASIC LABORATORY PRINCIPLES .................................................................................................................. 2
A. Laboratory Mathematics ................................................................................................................................... 2
B. Phlebotomy and Specimen collection ............................................................................................................... 2
PART II: CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES ........................................................................... 8
I. Carbohydrates ..................................................................................................................................................... 8
II. Lipids and Lipoproteins ................................................................................................................................... 12
III. Amino Acids and Proteins ............................................................................................................................... 18
IV. Non Protein Nitrogen compounds .................................................................................................................. 24
V. Enzymes ........................................................................................................................................................... 28
VI. Kidney Function Tests ..................................................................................................................................... 37
VII. Liver Function Tests ....................................................................................................................................... 38
VIII. Cardiac Profile ............................................................................................................................................... 40
XI. Electrolytes...................................................................................................................................................... 40
X. Blood gases and pH measurement .................................................................................................................. 46

XI. Trace Elements and Vitamins.......................................................................................................................... 48

PART III: ENDOCRINOLOGY ..................................................................................................................................... 50

PART IV: THERAPEUTIC DRUG MONITORING ......................................................................................................... 56
PART V: THE DRUGS OF ABUSE ............................................................................................................................... 60
PART VI: TOXICOLOGY............................................................................................................................................. 61

APPENDIX ................................................................................................................................................................ 64
REFERENCES ............................................................................................................................................................. 74
PART I: BASIC LABORATORY PRINCIPLES
A. Laboratory Mathematics- Concept of Solute and Solvent
A solution is a homogeneous mixture of one or more solutes dispersed molecularly in a sufficient
quantity of a dissolving solvent.

A.1. Expressing concentrations of solutions

A.1.a.Percent Solution: Percent implies parts per 100 and is independent of the molecular weight of a
substance.
Weight/Volume (w/v) : grams per 100 ml of diluent; most frequently used term for
percentsolution.
Weight/Weight (w/w)
Volume/Volume (v/v)
A.1.b. Molarity (M) 1. How many grams are needed to make 1 L
𝑚𝑎𝑠𝑠 (𝑔)
Mole = 𝑔𝑟𝑎𝑚 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔) of a 2 M solution of HCl?
2. Make up 250 mL of a 4.8 M solution of HCl.
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑚𝑜𝑙𝑒𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
Molarity of a solution = 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑙𝑖𝑡𝑒𝑟𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

A.1.c. Normality (N)

𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑔𝑟𝑎𝑚 𝑒𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
Normality of a solution =
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑙𝑖𝑡𝑒𝑟𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

𝑓𝑜𝑟𝑚𝑢𝑙𝑎 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔)

Gram equivalent weight =
𝑑𝑖𝑓𝑓𝑒𝑟𝑒𝑛𝑐𝑒 𝑖𝑛 𝑜𝑥𝑖𝑑𝑎𝑡𝑖𝑜𝑛 𝑠𝑡𝑎𝑡𝑒

A.1.d. Molality (m)

𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑚𝑜𝑙𝑒𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
Molality of a solution = 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑘𝑖𝑙𝑜𝑔𝑟𝑎𝑚𝑠 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡

A.1.e. Dilutions
*Dilution factor: ratio of concentrated or stock solution to the total solution volume; inverse
relationship to concentration.
- Simple dilutions: when you decide on the desired total volume and amount of stock to be used.

1. How do you make a 1:10 dilution of a serum

sample?
2.A 1:2 dilution of serum with saline had a
creatinine result of 8.6 mg/dL. Calculate the
actual serum creatinine concentration.

-Serial dilutions: multiple progressive dilutions ranging from more-concentrated solutions to less-
concentrated solutions.

*Dilution Ratio vs. Dilution Factor

𝑠𝑜𝑙𝑢𝑡𝑒 𝑠𝑜𝑙𝑢𝑡𝑒
Dilution Ratio: Dilution Factor:
𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑠𝑜𝑙𝑢𝑡𝑒+𝑠𝑜𝑙𝑣𝑒𝑛𝑡 (𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓𝑠𝑜𝑙′ 𝑛)
B.Phlebotomy and Specimen considerations
B.1. Specimen collection and handling

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 PROPER PATIENT IDENTIFICATION is the first step in specimen collection; the prime factor in order
to attain accurate results in clinical laboratory.
*two or three items of idenification should be used (Name, medical record number, date of birth, etc.)

B.1.a. General methods of blood collection:

The Vascular system

Bishop, Michael L., et.al. (2010). Clinical Chemistry – Principles, Procedures and Correlations. 6th ed. Lippincott Williams and Wilkins.

I. Arterial Puncture
- arterial blood is oxygenated and bright red in color; used for blood gas analysis and pH
measurement.

II. Venipuncture
- Venous blood is deoxygenated and dark red in color.

>Major veins for venipuncture:

*H pattern veins: 70% of population!
Median Cubital vein -located near or very center of antecubital fossa.
-prefered and first choice vein
-least painful site and generally safest to puncture
Cephalic vein -second choice vein
-usually only vein felt in obese patients
Basilic vein -third choice
-appear more accessible but located near the anterior and
posterior branches of the median cutaneous nerve.

>Venipuncture equipment:
1. Tourniquet - 1 minute recommended time limit of application
- applied 3-4 inches(10-15cm) above puncture site
- if blood pressure cuff is used inflate at 60 mmHg
2. Needle – 21 g standard fo venipunture
- length: 1 in o 1.5 in for 21 to 23 g
½ to ¾ in for butterfly needle
3. Evacuated tube system (ETS) – prefered venipuncture method

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 - basic components: multisample needle, tube holder, and various types
of evacuated tubes.

STOPPER COLOR ADDITIVE DEPARTMENT(S)

LIGHT BLUE Sodium citrate Coagulation
RED (GLASS) none Chemistry, bloodbank,
serology/immunology
RED (PLASTIC) Clot activator Chemistry
RED/LIGHT GRAY (PLASTIC) Nonadditive NA (Discard tube only)
RED/BLACK (TIGER) Clot activator and gel Chemistry
GOLD separator
RED/GOLD
GREEN/GRAY Lithium heparin and gel Chemistry
LIGHT GREEN separator
GREEN Lithium heparin Chemistry
Sodium heparin
LAVANDER EDTA Hematology
PINK Blood bank
GRAY Sodium fluoride and Chemistry
potassium oxalate
Sodium fluoride and EDTA
Sodium fluoride
ORANGE Thrombin Chemistry
GRAY/YELLOW
ROYAL BLUE None (red label) Chemistry
EDTA (lavander label)
Sodium heparin (green label)
TAN (GLASS TUBE) Sodium heparin Chemistry
TAN (PLASTIC) EDTA
YELLOW Sodium polyanethol Microbiology
sulfonate (SPS)
YELLOW Acid citrate dextrose (ACD) Blood
bank/immunohematology

4. Syringe system - 21- to 23-gauge, sterile, LuerLok style, hypodermic needles from 1 to 1 2/3 inches in
length

>Order of draw (evacuated tube and syringe)

Order of Draw Tube Stopper Color Rationale for Collection Order

Blood cultures (sterile collections) Yellow SPS Minimizes chance of microbial contamination
Sterile media bottle
Coagulation tubes Light blue The first additive tube in the order because all
other additives affect coagulation tests
Glass nonadditive tubes Red Prevents contamination by additives in other
tubes
Plastic clot activator tubes Red Filled after coagulationtests because silica
Serum separator tubes (SSTs) Red and gray rubber particles activate clotting and affect coagulation
Gold plastic
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 tests (carry-over of silica into subsequent tubes
can be overridden by anticoagulant in them)
Plasma separator tubes (PSTs) Green and gray rubber Heparin affects coagulation tests and interferes
Heparin tubes Light green plastic in collection of serum specimens causes the
Green least interference in tests other than
coagulation tests
EDTA tubes Lavander Responsible for more carry over problems than
Pink any other additive; elevates Na and K levels,
Plasma praparation tubes (PPTs) Pearl top chelates and decreases calcium and iron levels,
Oxalate/fluoride tubes Gray elevates PT and PTT results
Sodium fluoride and potassium oxalate affect
sodium and potassium levels, respectively, after
hematology tubes bacause oxalate damages cell
membranes and causes abnormal RBC
morphology. Oxalate interferes in enzyme
reactions.
!!! SITES TO BE AVOIDED!!!
1. intravenous line on both arms
2. burned or scarred areas
3. areas with hematoma
4. thrombosed veins
5. edematous arms
6. area of mastectomy
7.arms with AV shunt or fistula
8. 8. cast(s) on arm(s)

III. Skin puncture – capillary blood is a combination of venous blood and arterial blood
- Length of lancet : 1.75mm
- Depth of incision: <2.0mm for infants and children; <2.5mm for adults
*Prefered sites:
1. lateral plantar heal surface in newborn Order of filling micro collection tubes:
2. palmar surfaces of the 3rd and 4th fingers 1. EDTA
3. plantar surface of the big toe 2. Other tubes with additive
4. earlobes 3. nonadditive tubes

B.1.b. Specimen handling: Transport and Delivery

- transported to the laboratory in a plastic bag with a biohazard logo, a liquid-tight closure,
and a slip pocket for paperwork.
- Stat specimen: IMMEDIATELY!!!
- Routine specimen: delivered w/in 45 mins of collection and centrifuged w/in 1 hour of
arrival

B.2. Specimen Processing - complete clotting takes 30 to 60 minutes at room temperature

- Heparinized and other anticoagulated specimens centrifuged right away

B.3. Interferring conditions in measurement of analytes

Most common:
1. Hemolysis : causes dilutional effect
2. Bilirubin :Icteric sample at conc of 25.2 mg/L;causes spectral interference at 340nm & 500nm
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 3. Lipemia: triglyceride level of > 4.6mmol/L (400 mg/dl)

B.3.a.Pre-analytical variables contributing to variation in results

A. Controllable factors
1. Posture or position
> Lying to upright/standing/sitting position there is constriction of blood vessels and reduction of
plasma volume causing hemoconcentration to happen.
> Upright to supine causes the transfer of extravascular fluid to the vascular system bringing about
hemodilution of nondiffusable plasma constituents.

Lying to upright/standing/sitting Upright/standing/sitting to supine

Increase : Albumin, enzymes, calcium, BUN, Decrease:cholesterol, triglycerides and
iron,aldosterone lipoproteins.
*significant increase of potasium after 30 mins of *prolonged bed rest decrease plasma albumin due
standing to fluid retention

2. Exercise and Physical training

changes in concentrations of analytes due to
(1) shifts of fluid between the intravascular and interstitial compartments
(2) changes in hormone concentrations stimulated by the change in activity
(3) loss of fluid due to sweating

Increase Decrease
lactic acid, creatine, protein, CK, AST, lactate Cholesterol and triglycerides
dehydrogenase (LD), prolactin, testosterone, LH.
*ambulatory pxs: CK

3. Diurnal variation - pattern of production, excretion, and concentration of analytes each 24 hours
Increase Decrease
AM: ACTH, Iron(peaks PM: ACP, GH, PTH, TSH AM:Cortisol PM: ACTH, plasma
early to late morning), renin activity,
cortisol(peaks 4-6am), aldosterone
aldosterone, Prolactin

4. Travel – effect on normal circadian rythym

Increase Decrease
Urine catecholamine, serum glucose and cortisol
triglyceride.
*prolonged flight: fluid and sodium retention

5. Fasting
Require fasting: Fasting blood sugar, glucose tolerance test, triglycerides, lipid panel, gastrin,
insulin, aldosterone/renin.

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6. Diet : Recent food ingestion
Increase Decrease
Glucose, insulin, triglycerides, gastrin, and ionized chloride, phosphorus, potassium, amylase, and
calcium ALP
*caffeine: inc glucose *Malnutrition: dec total serum protein, albumin,
β-globulin, BUN, and creatinine.

7. Smoking - extent of the effect is related to the number of cigarettes smoked and to the amount of
smoke inhaled.
Increase Decrease
Catecholamines, cortisol, glucose, GH, cholesterol, Vitamin B12
triglyceride, ammonia, urea, lactate, insulin,
urinary 5-HIAA and plasma nonesterified fatty
acid.

8. Alcohol ingestion
Increase Decrease
Urate, triglycerides and GGT Glucose: hypoglycemia in chronic alcoholism

9. Use of recreational drugs

- Morphine: increase activity of amylase, lipase, ALT, AST, ALP, and concentration of serum bilirubin.
decrease insulin, norepinephrine, pancreatic polypeptide, and neurotensin
- Cannabis: increase sodium, potassium, urea, chloride, and insulin,
Decrease creatinine, glucose, and urate
- Diuretics decrease plasma sodium and potassium

10. Stress/ Anxiety

Increase: ACTH, Cortisol, catecholamine, prolactin, insulin, albumin, glucose and lactate.

11. Tourniquet application

>Recommended time of application: 1 minute
> Prolonged: Hemoconcentration
> Prolonged use with fist clenching: Increase serum potassium

B. Uncontrollable factors: Physiologic variables

1. Age: Albumin, ALP, phosphorus,and cholesterol

2. Gender
Increase in Male Increase in Female
Albumin, ALP, creatine, Calcium, uric acid, Iron, cholesterol, gamma globulins, and alpha
creatinine kinase, aspartate aminotransferase, lipoproteins(HDL)
phosphate, BUN, magnesium,bilirubin,a nd
cholesterol

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3. Race
- Total protein ↑ (black), albumin ↓ (black); IgG 40% ↑, and IgA 20%↑ (black male vs. white male); →
CK/LD ↑ black males; ↑ cholesterol and triglycerides > white >40 years old (glucose-incidence diabetes
in Asian, black, Native American, Hispanic)

Require ice: Lactic acid, ammonia, blood gas (if not cooled = ↓ pH, and po2) (immediate cooling)

Hemolysis ↑ K, ammonia, PO4, Fe, Mg2, ALT, AST, LD, ALP, catecholamines, CK (marked hemolysis)

*POINT OF CARE TESTING (POCT): those analytical patient-testing activities provided within the
institution, but performed outside the physical facilities of the clinical laboratories –CAP
-Nursing, perfusion, or respiratory therapy staff or resident or attending physicians may perform POCT

PART II: CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES

I. CARBOHYDRATES- compounds containing C, H, and O

-aldehyde or ketone derivatives of polyhydroxy (more than one OH group) alcohols,
or compounds that yield these derivatives on hydrolysis
-metabolism generates pyruvic acid, lactic acid and acetylcoenzyme A as
intermediate products
>Classification:
1. According to number of carbon atoms
a. Monossacharides- simple sugars containing either three, four, five or six carbon atoms
- most common hexoses: Glucose, Fructose & Galactose
d. Dissacharides - two monosaccharides joined covalently by an 0-glycosidic bond, with the loss
of a molecule of water
Maltose = glucose + glucose
Lactose = glucose + galactose
Sucrose = glucose + fructose
c. Polyssacharides - linkage of multiple monosaccharide units
- major storage of carbohydrates: STARCH (plants) and GLYCOGEN (animals)
- Starch is a mixture of amylose and amylopectins
- structure of glycogen is similar to amylopectin,but branching is more extensive
and occurs every 8 to 12 glucose residues
d. Glycoproteins – integral membrane proteins that has oligosaccharide covalently attached to
extracellular region

2. According to reducing property

• Reducing sugars: glucose, maltose, fructose, lactose and galactose
• Non-reducing sugar: sucrose

>Regulation of blood glucose concentration : the role of the pancreas as an endocrine and exocrine organ

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Pathways in glucose metabolism

Glycolysis Metabolism of glucose molecule to pyruvate or

lactate for production of energy
Gluconeogenesis Formation of glucose-6-phosphate from
noncarbohydrate sources
Glycogenolysis Breakdown of glycogen to glucose for use as
energy
Glycogenesis Conversion of glucose to glycogen for storage
Lipogenesis Conversion of carbohydrates to fatty acids
Lipolysis Decomposition of fat

a. Exocrine- production and secretion of amylase for breakdown of ingested complex carbohydrates
b. Endocrine- secretion of hormones:
1. Insulin:synthesized by Beta cells of the pancreas; for entry of glucose in to the cell;
onlyHYPOGLYCEMIC AGENT.
Promotes: glycogenesis, lipogenesis and glycolysis
Decreases: glycogenolysis
2.Glucagon: synthesized by Alpha cells of the pancreas; released during stress and fasting states;
Primary HYPERGLYCEMIC AGENT.
Promotes: Glycogenolysis
3. Somatostatin : synthesized by Delta cells of the pancreas; INHIBITS action of insulin, glucagon
and growth hormone.

>Clinical significance: Condition of carbohydrate metabolism

1. Hyperglycemia: FBS level ≥126 mg/dl
Renal threshold for glucose: 140-160 mg/dl or 160-180 mg/dl
CRITICAL VALUE: >500mg/dl
Causes: stress, severe infection, dehydration, pregancy, pancreatectomy, hemochromatosis,
insulin deficiency or abnormal insulin receptor.
Laboratory findings: electrolyte imbalance (↓Na and HCO3,↑ K), acidosis, increase urine
specific gravity, increase glucose and presence of ketones in plasma and urine.

2. Hypoglycemia: imbalance between glucose production and utilization

Diagnosis based on Whipple’s Triad: low blood glucose conc, typical signs and symptoms
alleviated by glucose administration
• Hypoglycemic values:
a. 65 to 70 mg/dl: hormonal response- release of glucagon
b. <60 mg/dl: suggestive of hypoglycemia
c. 50-55mg/dl: observable signs and symptoms
d.40 mg/dl: CRITICAL VALUE
e. 20-30 mg/dl:severe CNS dysfunction

DIABETES MELLITUS
- group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion,
insulin action, or both. FBS of ≥126 mg/dl on more than one testing are diagnostic of DM
- ratio of β-hydroxybutyrate to acetoacetate in severe DM is 6:1
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>Classification:
TYPE 1 Diabetes Mellitus TYPE 2 Diabetes Mellitus
-Insulin Dependent Diabetes Mellitus (IDDM) - Non-insulin dependent Diabetes Mellitus
-juvenile onset Diabetes Mellitus; Ketosis prone - Adult onset DM
diabetes - Receptor-deficient Diabetes Mellitus
-autoimmune Beta cell destruction - resistance to insulin
-genetic association with HLA DR3 and DR4 -risk factors: genetics obesity sedentary lifestyle,
-autoantibodies: glutamic acid decarboxylase PCOS, dyslipidemia and hypertension
(GAD65) and insulin autoantibodies (IAAA), Islet - 90-95% incidence rate
cell autoantibody, and Tyrosine phosphatase IA-2 - detectable C-peptide levels
and IA-2B autoantibodies - oral medication and lifestyle (weight loss)
-5% to 10% incidence rate changes
- very low undetectable C-peptide levels
- no therapy available
*C-peptide is formed during the conversion of pro-insulin to insulin; circulating amount is a reliable
indication for pancreatic and insulin secretions (beta cell function).
- primary indication for measurement of C-peptide is the evaluation of fasting hypoglycemia; When
hypoglycemia is due to surreptitious insulin injection, insulin concentrations are high but C-peptide
concentrations are low.

*Gestational Diabetes Mellitus – glucose intolerance during pregnancy due to metabollic and hormonal
changes
- Screening is done between 24 and 28 weeks of gestation
- Increases risk of subsequent diabetes particulary type 2 diabetes

>Diagnostic criteria for GDM

1. FBS ≥92 mg/dl
2. 1-hour GCT ≥180mg/dl
3. 2-hour OGTT ≥153 mg/dl

>Glucose Methodologies
- Fasting glucose in whole blood is 11% to15% lower than in serum or plasma
- serum is separated from cells within 30 minutes
- venous blood glucose is 7mg/dl lower than capillary blood glucose
-CSF glucose is 60% of the plasma concentration
- fasting blood glucose increase by 2mg/dl per decade as a person age
- glycolysis lowers glucose level by 7mg/dl/hr at room temperature and by 2mg/dl/hr at
refrigerator temperature

Samples for glucose measurement

1. Random blood sugar (RBS)
2. Fasting blood sugar (FBS) : for measurement of overall glucose homeostasis; at least 8 hours
fasting
3. 2-hour Post prandial blood sugar: for glucose metabolism
4. Glucose tolerance test (GTT)
I. Chemical Methods III. Glucose Tolerance test

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 A. Oxidation reduction method a. Oral glucose tolerance test (OGTT): to detect
1. Alkaline copper reduction gestational Diabetes Mellitus
a. Folin Wu - One step approach for high risk
b. Nelson Somogyi individuals
c. Neocuprine - Two step approach for average risk
d. Benedict’s women
2. Alkaline ferric reduction (Hagedorn Jensen) b. Intravenous glucose tolerance test (IVGTT):
B. Condensation method: Dubowski – Ortho for DM patients with gastrointestinal disorder
toluidine
*prior to OGTT, patient must have
unrestricted diet of 150g carbohydrates for 3
days
*Glucose load
75 g WHO standard load
1.75g/kg body weight for children
II. Enzymatic method IV. Measure of Glycemic control
1. Hexokinase: most accurate and reference A. Glycosylated Hemoglobin (HbA1C):glucose
method for glucose molecule attached to one or both N-terminal
gluocose + ATP hexokinase glucose 6-PO4 +ADP valines of the βpolypeptide chains of normal
glucose 6-PO4 + NADP G-6-PD NADPH + H+ + 6- adult hemoglobin
phosphogluconate - Reliable in monitoring glucose control in the
previous 2-4 months.
2. Glucose Oxidase: specific for β-D glucose - specimen: EDTA whole blood
- Affinity chromatography prefered method of
glucose + O2 + H2O2 glucose oxidase gluconic acid + H2O2 measurement
H2O2 + reduced chromogen peroxidase oxidized
chromogen + H2O2 5.7% - 6.4% : increased risk for diabetes (pre-
diabetes)
*Mutarotase converts α-D-glucose to β-D-glucose. ≥6.5%: on at least two ocassions- inidcative of
*Polarographic glucose oxidase: Oxygen DM
consumption is proportional to glucose *for every 1% change in HbA1c value,
concentration. 35mg/dl is added to plasma glucose.
3. Glucose Dehydrogenase: Close agreement with
hexokinase procedures B. Fructosamine : Glycosylated albumin
- reflection of glucose control over the previous
3-6 weeks
-recommended for diabetic patients with
shortened red cell life span

Criteria for DM diagnosis

RBS and 2hrPP ≥200mg/dl (11.1 mmol/L)
FBS ≥126mg/dl (7.0 mmol/L)
HbA1c ≥6.5%
OGTT ≥200mg/dl in 2hr plasma glucose

Impaired Fasting blood sugar: 100-125 mg/dl

Impaired glucose tolerance: 140-199 mg/dl
GLYCOGEN STORAGE DISEASE

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*Von Gierke is the most common Glycogen storage disease, and is associated with hyperlipidemia

II. LIPIDS and LIPOPROTEINS

- Serve as hormones, energy source, aids in digestion, and act as cellular components in cell
membrane of man.
- involved in the development of atherosclerosis, a pathogenic process that is the underlying cause
of the common cardiovascular disorders of myocardial infarction,cerebrovascular disease, and
peripheral vascular disease
- insoluble in blood and water, but soluble in organic solvents such as chloroform and ether
- commonly refered to as fats;composed of mostly carbon-hydrogen (C-H) bonds
- fat soluble Vitamins A,D,E,K

>Basic Lipids
A. Cholesterol
- found almost exclusively in animals and is a key membrane component of all cells and bile acids
- steroid alcohol with 27 carbon atoms that are arranged in a tetracyclical sterane ring system, with a
C-H side chain
- amphipathic lipid and is found on the surface of lipid layers along with phospholipids
- precursor of five major steroid: progestins, glucocorticoids, mineralocorticoids, androgens and
estrogens
- evaluates risk for atherosclerosis, myocardial and coronary arterial occlusion

>Forms of Cholesterol
1. Cholesteryl ester – 70%
- hydroxyl group conjugated by an ester bond to a fatty acid
- excess cholesterol in cells is esterified by acylcholesterol acyltransferase (ACAT)
- esterification in circulation is facilitated by lecithin cholesterol acyltranserase(LCAT)
- LCAT enables HDL to accumulate cholesterol as cholesterol ester

2. Free Cholesterol – 30%

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 - polar nonesterified alcohol
- found in plasma, serum and RBCs

* Cholesterol catabolism

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B. Fatty acids
- linear chains of carbon-hydrogen bonds that terminate with a carboxyl group
- classified as short chain (2-4 carbon atoms), medium chain (6-10 carbon atoms), or long chain(12 -
26 carbon atoms)
- saturated fatty acids have no double bonds between their carbon atoms, monounsaturated fatty
acid have one double bond, while polyunsaturated fatty acids have multiple bonds between their
carbon atoms
- mostly are constituents of phospholipids and triglycerides
- provide substance for conversion to glucose (gluconeogenesis)

C. Triglycerides (Neutral fats)

- contains 3 molecules of fatty acid and one molecule of glycerol by ester bonds
- main storage lipid in man (adipose tissue)
- has no charge so classified as neutral lipid
- breakdown is facilitated by lipoprotein lipase (LPL), epinephrine, and cortisol.
- Evaluates suspected atheroscleosis and measures the body’s ability to metabolize fat

D. Phospholipids (conjugated lipid)

- similar in structure to triglycerides except that they only have two esterified fatty acid
- serve as surfactant by altering fluid surface tension to prevent alveolar collapse during expiration
- forms: Lecithin/Phosphatidyl choline (70%), Spingomyelin ( 20%), and Cephalin (10%)
E. Lipoproteins

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- spherical particles with nonpolar neutral lipids (triglycerides and cholesterol esters) in their core
and more polar amphipathic lipids (phospholipids and free cholesterol) at their surface
- contain one or more specific proteins, called apolipoproteinswhich aid in solubilization of lipids
- carries cholesterol and triglycerides travelling in the circulation

➢ Major Liporproteins
1. CHYLOMICRONS (CM) - largest and least dense; produced by the
intestine that transport lipids of dietary
(exogenous) origin to the tissues of the body
- produce a “milky” plasma in large
concentration
2. VERY LOW DENSITY LIPOPROTEIN (VLDL) - Pre-beta lipoprotein
- transports endogenous Triglyceride from liver
to muscle and peripheral tissues
- produce “turbid” plasma when present in large
amounts
3. HIGH DENSITY LIPOPROTEIN - Alpha lipoprotein
- smallest but most dense
- transport excess cholesterol from tissues to the
liver (REVERSE CHOLESTEROL TRANSPORT)

4. LOW DENSITY LIPOPROTEIN (LDL) - Beta Lipoprotein

- major end product from catabolism of VLDL
- most cholesterol rich and most atherogenic

Characteristics Chylomicrons VLDL LDL HDL

Density (g/ml) <0.93 0.93-1.006 1.019-1.063 1.063-1.21
Total lipid (% by 98 89-96 77 50
weight)
Triglyceride (% by 84 44-60 11 3
weight)
Total cholesterol 7 16-22 62 19
(% by weight)

SIGNIFICANT HUMAN APOLIPOPROTEINS

Apolipoprotein Plasma concentration Major Lipoprotein Function

(mg/dL) location
Apo A-I 100-200 HDL Structural, LCAT
activator, ABCA1 lipid
acceptor
Apo A-II 20-50 HDL Structural
Apo A-IV 10-20 Chylomicrons, VLDL, Structural
HDL
Apo B-100 70-125 LDL, VLDL Structural, LDL
receptor ligand

14
 Apo B-48 <5 Chylomicrons Structural, remnant
receptor ligand
Apo C-I 5-8 Chylos, VLDL, HDL Structural
Apo C-II 3-7 Chylos, VLDL, HDL Stuctural, LPL cofactor
Apo C-III 10-12 Chylos, VLDL, HDL Sturctural, LPL
inhibitor
Apo E 3-15 VLDL, HDL Structural, LDL
receptor ligand
Apo(a) <30 Lp(a) Structural,
plasminogen inhibitor

*Additional
> Apo J : cell aggregating factor in sertoli cells ; involved in apoptosis; also known as clusterin
> Apo F : regulates CETP function
> Apo M : linked to HDL remodeling
> Apo E : associated with higher risk for alzheimer’s
>Apo (a) : homologous to plasminogen

➢ Minor Lipoproteins
a. Intermediate lipoprotein (IDL) – VLDL remnant- product of VLDL catabolism
b.Lipoprotein (a)/ Lp (a)- Sinking pre-beta lipoprotein

> Abnormal lipoproteins

a. Lipoprotein X – found in obstructive jaundice and LCAT deficiency; sensitive indicator of
cholestasis
b. β-VLDL- floating beta lipoprotein; VLDL rich in cholesterol

>Lipid methodologies
- Basic lipids that are measured in the laboratory include cholesterol, triglcerides, HDL cholesterol, and
LDL cholesterol

A. Cholesterol assays
1. Enzymatic methods : assay of choice for routine cholesterol measurement; measure total cholesterol
(TC) directly in plasma or serum

1. Cholesteryl ester + H2O cholestryl esterease Cholesterol + Fatty acid

Cholesterol oxidase
2. cholesterol + O2 Cholestenone + H2O2
3. H2O2 + Dye Peroxidase Color

The intensity of the resulting color, proportional to the amount of cholesterol, can be measured by a
spectrophotometer, usually at a wavelength around 500 nm
*Interference: elevated ascorbic acid level in plasma results to falsely decrease Total Cholesterol

2. Chemical methods : employs dehydration and oxidation of cholesterol to form a colored compound
2.a. Liebermann Burchardt reaction : uses acetic anhydride; end product is a green color
Color developer(Lieberman Burchardt reagent): Glacial acetic acid, acetic
anhydride,and concentrated H2SO4

15
 2.b. Salkowski : uses ferric chloride; end product is red color
GENERAL METHODS
One Step Colorimetry (Pearson, Stern and Mac Gavack)
Two Step Extraction + Colorimetry ( Bloor’s)
Three Step Saponification + Extraction + Colorimetry (Abell-
Kendall)
Four Step Saponification + Extraction + Colorimetry +
Precipitation ( Schoenheimer Sperry, Parekh and
Jung)

B.Triglyceride assays :
1. Enzymatic methods : relatively specific, rapid and easy to use; linear in the concentration range up
to about 700 mg/dL (7.91 mmol/ L)

1. Triglyceride + H2O2Bacterial lipase Fatty acid + Glycerol

2. Glycerol + ATP Glycerokinase Glycerophosphate +ADP
3. Glycerophosphate + O2glycerophosphate oxidase Dihydroxyacetone + H2O2
4. H2O2 + Dye Peroxidase Color
2. Chemical methods
2.a. Colorimetric Method (Van Handel & Zilversmith) :end product is blue in color
2.b. Fluororimetric Method (Hantzsch condensation) : end product is diacetyl lutidine

>CDC Reference method (Modified Van Handel and Zilversmith method)

-involves alkaline hydrolysis, solvent extraction, and a color reaction with chromotropic acid.

C. Lipoprotein methodologies : differentiated based on electrophoresis and buoyant density

(ultracentrifugation)
- prefered sample is a blood placed in a tube with serum separator
- EDTA plasma is prefered for electrophoresis and ultracentrifugation

1. Ultracentrifugation
- reference method for quantitation of LPPs; best means to compare LPP classes
- expressed in svedberg units (s)
- VLDL and CM are the most lipid rich thus they are the most buoyant, followed by LDL and lastly
by HDL which has the least lipid content

2. Electrophoresis
- agarose gel is the most commonly used support media for LPP electrophoresis

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3. Polyanion precipitation methods
- uses polyanions such as heparin sulfate, dextran sulfate, phosphotungstate in the presence of divalent
cations such as calcium, magnesium and manganese.
- the more dissimilar the LPPs are from one another, the better is the separation.
-HDL uses dextran sulfate with magnesium ( precipitant)
4. Standing Plasma Test
- for the detection of Chylomicrons
- a 2ml plasma is allowed to stand overnight at ref temp (40C) and if CM is present in appreciable amount,
then a floating creamy layer may be observed

Friedwald Calculation for LDL cholesterol determination

LDC cholesterol = Total cholesterol – HDL cholesterol – VLDL

𝑃𝑙𝑎𝑠𝑚𝑎 𝑇𝑟𝑖𝑔𝑙𝑦𝑐𝑒𝑟𝑖𝑑𝑒
VLDL (mmol/L) =
2.175

𝑃𝑙𝑎𝑠𝑚𝑎 𝑇𝑟𝑖𝑔𝑙𝑦𝑐𝑒𝑟𝑖𝑑𝑒
VLDL (mg/dl) =
5.0

➢ Disorders associated with Lipids and Lipoproteins

1. Abetalipoproteinemia ( Bassen-Kornzweig Syndrome) - autosomal recessive; DEFECTIVE APO B
SYNTHESIS
2. Lipoprotein Lipase(LPL) deficiency - results in inability to clear Chylomicrons
3. LCAT deficiency – mutation in LCAT gene

> Fredrickson Classification of Hyperlipidemias

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mia&gs_l=img.3...1393659.1440468.0.1440664.63.50.4.0.0.0.742.7948.0j2j7j2j3j4j2.20.0....0...1c.1.64.img..39.10.3496...0j0i67k1j0i10k1j0i5i10i30k1j0i5i30k1j0i10i24k1j0i24k1.0.Dt86xQ8pylw#imgrc=ShVVMmXeV7EsSM:

17
 ➢ Lipid Storage Diseases

Disease Enzyme deficiency

1. Fabry’s α-galactosidase
2.Gaucher’s β-galactosidase
3. Krabbe Cerebroside β galactosidase
4. Niemann Pick Spingomyelinase
5. Sandhoff Total Hexosaminidase
6. Tay Sach Hexosaminidase A
7. Metachromatic Leukodystrophy Arylsulfatase A

LIPID NCEP GUIDELINES FOR NORMAL VALUES

LDL (mg/dL) HDL (mg/dL) TAG (mg/dL) TC (mg/dL)

Desirable <100 ≥60 <150 <200
Borderline high 130-159 150-199 200-239
High 160-189 200-499 ≥240
Very high ≥190 ≥500
low <40

III. AMINO ACIDS AND PROTEINS

A. Amino acids – building blocks of proteins; chemical properties of the amino acids of proteins
determine the biologic activity of the protein

B. Proteins - Any of a group of complex organic compounds that contain carbon, hydrogen, oxygen,
nitrogen, and usually sulfur (the characteristic element being nitrogen)and are distributed widely in
plants and animals
- the backboned of all proteins is a continuous chain of carbon and nitrogen atoms joined
together through peptide bonds between adjacent amino acids
- most plasma proteins are synthesized by the liver except for immunoglobulins which are
synthsized in plasma cells

> Structure
a. Primary : represents the number and types of amino acids in the specific amino acid
sequence; the linear sequence of the amino acid
- it determines the identity of a protein
b. Secondary : refers to specific regular three-dimensional conformation into which portions of
the polypeptide chain fold.
- the three structures are α-helix, β-pleated sheet and the bend conformation
c. Tertiary : actual three-dimensional structure or folding pattern of the protein which is
determined by its amino acid sequence
- confers physical and chemical properties of the protein
d. Quaternary : shape or structure that results from the interaction of more than one protein
molecule, or protein subunits, held together by noncovalent forces such as hydrogen bonds and
electrostatic interactions, which are part of the larger protein complex with a precise three-
dimensional configuration

18
➢ Notes to Remember!!!!
- Isoelectric point : pH at which amino acid or protein has no net charge
- Proteins can function as enzymes, hormones, transport proteins, immunoglobulins(antibodies),
structural and storage proteins, energy source and maintains water distribution (osmotic force)
- Simple proteins contain peptide chains composed of only amino acids
- Conjugated proteinsconsist of a protein and a nonprotein (prosthetic) group.

➢ The Plasma Proteins

1. Prealbumin (Transthyretin) – a small protein that can bind a molecule of thyroxine, thus it is also
called as thyroxine-binding prealbumin (TBPA) ; it can also bind and transport retinol (Vitamin A)
- sensitive marker for poor nutrintional status (malnutrition)
- landmark that confirms that a sample is really CSF

2. Albumin – most abundant protein in normal plasma

- responsible for colloid osmotic pressure of the intravascular fluid
- serve as a buffer for blood, transports a number of molecules and is a negative acute phase
reactant
- also an indicator of nutritional status
3. Globulin : group of proteins consists of α1, α2,β,and γ fractions
- measurement: Total protein – Albumin = Globulin

α1globulin α2 globulin Β globulin γ globulin

Alpha1-antitrypsin, Alpha2- Beta2 microglobulin, Immunoglobulins
aplha1-acid macroglobulin, Ferritin, hemopexin, *CRP
glycoprotein, alpha1 ceruloplasmin and complements,
antichymotrypsin, haptoglobin fibrinogen, LDL, VLDL
AFP, alpha-lipoprotein and CRP
and Gc globulin

MOST OF:
Alpha1 globulins is alpha1 antitrypsin
Alpha2 globulins is alpha2 macroglobulin
Beta globulins is Transferrin

a. alpha1-antitrypsin (AAT) : a serpin – serine proteinase inhibitor

- major inhibitor of protease activity
- deficiency leads to emphysema

b. alpha1-fetoprotein (AFP) : synthesized in the developing embryo and fetus and then by the
parenchymal cells of the liver
- Elevated maternal serum or amniotic fluid AFP indicates the possibility of an open neural tube
or abdominal wall defect in the fetus

c. alpha1-acid glycoprotein / Orosomucoid (AAG) :major plasma glycoprotein which is negatively

charged even in acid solutions
- an acute phase reactant
- levels in the serum provides a useful diagnostic tool in neonates with bacterial infection
19
d. alpha1 antichymotrypsin : member of the serpin family; inhibits the activity of the enzymes
cathepsin G, pancreatic elastase, mast cell chymase, and chymotrypsin by cleaving them into a
different shape
e. Gc globulin (Group-specific component; Vitamin D binding protein) : major carrier protein of
vitamin D and its metabolites in the circulation and also transports components such as fatty acids
and endotoxin

f. Haptoglobin : binds free hemoglobin irreversably

-testing is used primarily to help detect and evaluate hemolytic anemia and to distinguish it
from anemia due to other causes
- can be quantitated in terms of its hemoglobin-binding capacity or by immunologic means such
as nephelometry

g. Ceruloplasmin : a copper binding protein; serves as an acute phase reactant

- primarily ordered along with blood and/or urine copper tests to help diagnose Wilson’s
disease (autosomal recessive inherited disorder associated with decreased levelstypically 0.1
g/L) of ceruloplasmin and excess storage of copper in the liver, brain, and other organs)

h. alpha2 macroglobulin : largest major nonimmunoglobulin protein in plasma

- concentration rises 10x in nephrotic syndrome; also rises in diabetes and liver disease
- inhibits proteases such as trypsin, pepsin and plasmin

i. Transferrin (siderophilin) : transports ferric ions from the iron stores of intracellular or mucosal
ferritin to bone marrow
- levels are tested for to determine the cause of anemia, to gauge iron metabolism, and to
determine the iron-carrying capacity of the blood
- transferrin will be abnormally high in iron deficiency anemia; transferrin deficiency will result
to accumulation of iron in apoferritin or histiocytes and may precipitate as hemosiderin

j. Hemopexin : functions to scavenge the heme released or lost by the turnover of heme proteins
like hemoglobin; binds heme with the highest affinity of any known protein
- used for diagnosis of early hemolysis

k. Beta2 microglobulin : light chain component of the major histocompatibility complex (HLA)
-found on the surface of most nucleated cells and is present in high concentrations on
lymphocytes

l. Complement : one of the natural defense mechanisms that protects the human body from
infections
- C3 is the most abundant complement protein in plasma followed by C4

m. Fibrinogen : one of the largest protein in plasma; classified as glycoprotein

- most abundant of the coagulation factors

n. C-reactive Protein : first to rise in cases of inflammation

- its name originates from its ability to bind to the C-polysaccharide of the pneumococcus

20
 o. Immunoglobulins : are glycoproteins composed of 82%–96% protein and 4%–18% carbohydrate
produced by white blood cells, known as B cells, that confer humoral immunity

➢ Other Proteins of Importance

a. Myoglobin : primary oxygen carrying protein found in striated and cardiac muscle
- serves as a cardiac biomarker together with troponin to detect heart attack
-it is a potential nephrotoxin

b. Troponins : complex of three regulatory proteins that bind the thin filaments of cardiac muscle
(actin and myosin)
- Cardiac Troponin (cTn) is the gold standard for diagnosis of AMI
- measured on serum or heparinized plasma by ELISA or immunoenzymometric assay

c. B-type natriuretic peptide (BNP) : serve as cardiac marker; increase in response to systolic and
diastolic dysfunction

d. Cystatin C : a cysteine proteinase inhibitor; proposed as alternate test for serum creatinine and
creatinine clearance test for evaluation of kidney function

➢ Total Protein Abnormalities

A. Hypoproteinemia : occurs when there is a negative nitrogen balance
- one cause of low protein concentration in blood is due to excessive loss through urine in cases
of renal disease
- other causes include decreased intake (malnutrition), intestinal malabsorption (sprue) , loss
through wounds and due to increased protein catabolism seen in burns and traumas.

B. Hyperproteinemia : not an actual disease rather a result of an underlying condition which is

dehydration
- excsessive production of gamma globulins such as in multiple myeloma, is another condition
that cause increased blood protein concentration

Total Protein Albumin Globulin Disease

Normal, decreased decreased increased Hepatic damage
- Cirrhosis β-γ
bridging
- Hepatitis inc
gamma-globulins
- Obstructive
jaundice inc α2, β-
globulins
Burns
Infections
- Acute inc α1, and
α2-globulins
- Chronic inc α1,
and γ globulin

21
 decreased decreased normal Malabsorption
Inadequate diet
Nephrotic syndrome
inc α2, β-globulins, dec
γ-globulins
decreased normal decreased Immundeficiency
syndrome
decreased decreased decreased Salt retention
syndrome
increased increased increased Dehydration
increased normal Increased Multiple myeloma
Monoclonal and
polyclonal
gammopathies

➢ Methods of Analysis
1. Total Nitrogen deterimination : measures all chemically bound nitrogen in the sample; can be
performed in plasma and urine
- method uses chemiluminiscence; useful in assessing nitrogen balance

2. Total Protein : fasting specimen may not be required

> Refernce interval : 6.5–8.3 g/dL (65–83 g/L) for ambulatory adults
6.0–7.8 g/dL (60–78 g/L)in recumbent position

METHOD PRINCIPLE COMMENTS

Kjeldahl Digestion of protein with Reference method
subsequent measurement of Assume average nitrogen
nitrogen content content is 16%
- Digestion is carried out
with the use of TCA or
tungstic acid
- Ammonium ion produced
through digestion is then
measured through
alkalinization, distillation,
acid titration or
nesslerization
Biuret Formation of violet colored Routine Method : Most
chelate between copper ions widely used
and peptide bonds - RECCOMENDED by IFCC
- Requires at least two
peptide bonds and an
alkaline medium
Refractometry Measurement of refractive Rapid and simple; assume
index due to solutes in serum nonprotein solids are present
in same concentration as the
calibrating serum

22
 Dye Binding Protein binds to dye and Mainly used in research
causes a spectral shift in - Bromphenol blue,
absorbance maximum of the Ponceau S, amido black
dye 10B, lissamine green, and
Coomassie brilliant blue
have been used to stain
protein bands after
electrophoresis

3. Albumin determination : dye binding methods are the most widely used; the pH of the solution
is adjusted so that albumin is positively charged
- amount of albumin is calculated by measurement of the absorbance of the albumin-dye
complex

Method Principle Comment

Salt precipitation Globulins are precipitated in Labor intensive
high salt conc; albumin in
supernatant is quantitated by
biuret reaction
Electrophoresis Separation according to Accurate; gives overview of
electric charge relative changes in different
protein fractions
Dye binding
Methyl orange Nonspecific for albumin
HABA (2,4 – hydroxybenzene- Albumin binds to dye; causes Many interferences
benzoic acid) shift in absorbance maximum (salicylates, bilirubin)
BCG (bromcresol green) Sensitive; overestimates low
albumin levels; most
commonly used dye
BCP (bromcresol purple) Specific, sensitve and precise

4. Total Globulins : level in serum is determined by a direct colorimetric method using glyoxylic acid
-Glyoxylic acid, in the presence of Copper and in an acid medium (acetic acid and H2SO4),
condenses with tryptophan found in globulins to produce a purple color

5. Electrophoresis : usually performed when an abnormality in total protein or albumin is observed

- it separates proteins on the basis of their electrical charges densities
- movement will be determined by the pH of the surrounding buffer
- at a pH greater than the isoelectric point, proteins will be negatively charged; a pH of less than
the isoelectric point will render a positive charge to the proteins
- cations (positive net charge) migrate to the cathode (negative terminal), whereas anions
(negative net charge) migrate to the anode (positive terminal)
-Cellulose acetate and agarose gel are the supporting media commonly used in the clinical
laboratory
-Monoclonal immunoglobulin diseaseis the most significant finding in an electrophoretic
pattern
23
 !!STAINS!!
Bromcresol green : most common
Bromcresol purple : most sensitive to albumin
Ninhydrin : for amino acids
Tetrabromophenol : urine reagent strip
• Prealbumin migrate towards the anode before albumin
• Gamma globulins remains at the origin
• Beta-gamma bridging seen in liver cirrhosis
• Increased A2M, decreased albumin in nephrotic syndrome
• Increased in beta fraction when plasma instead of serum is used
• Decreased albumin, increased alpha1 and alpha2 proteins in acute inflammation
• Decreased A1 antitrypsin seen in emphysema
• CSF oligoclonal banding (two or more IgG bands in gamma region) seen in multiple sclerosis
*in Multiple sclerosis oligoclonal banding is seen in CSF but not in serum

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➢ Aminoacidopathies
o Alkaptonuria : absence of homogentisate oxidase; urine darkens upon standing
o Homocystinuria: impaired activity of cystathione B-synthetase; screened by Modified
Guthrie test
o Maple syrup urine disease (MSUD) : reduced or absence of a-ketoacid decarboxylase
▪ Leads to accumulation of brached-chain amino acid –leucine, isoleucine and valine
▪ Screening test: Modified Guthrie test using 4-azaleucine as antagonist
o Phenylketonuria : reduced phenylalanine hydrolase
▪ Screening test: Guthrie bacterial inhibition assay using bacillus subtilis spores
▪ Positive result is presence of bacterial growth

IV. NONPROTEIN NITROGEN COMPOUNDS (NPN)

- Traditionally has been used to monitor renal function

A. UREA – NPN present in highest concentration in blood; the major excretory product of protein and
amino acid catabolism
- 90% is excreted through the kidneys; it is neither actively reabsorbed nor secreted by the
tubules but is filtered freely by the glomeruli
- measurement in blood is used to evaluate renal function, to assess hydration status, to
determine nitrogen balance, to aid in the diagnosis of renal disease, and to verify adequacy of
dialysis
- Blood Urea Nitrogen (BUN) concentration can be converted to Urea concentration by

24
 multiplying 2.14 --- BUN x 2.14 = Urea concentration

➢ ANALYTICAL METHODS
1. Chemical method : FEARON reaction
- condensation of diacetyl with urea to form the chromogen diazine which is yellow in color
! inexpensive and lacks specificity

2. Enzymatic Method: ammonia formation

! greater specificity but more expensive
a. urea hydrolysis by UREASE (* fluoride or citrate will inhibit urease)
- urease is prepared from jackbeans
- hydrolysis is done as a preliminary step in all other enzymatic methods
- ammonia generated from hydrolysis of urea is quantitated by the Berthelot reaction and
enzymatic reaction with glutamate dehydrogenase
- 8-23 mg/dL (2.9 – 8.2 mmol/L)

Urease
Urea + 2H2O 2NH4+ + CO32- NH4+ + 2-oxoglutarate GLDH glutamate + H2O
NADH + H NAD+
3. Isotope dilution mass spectrometry (IDMS) : proposed!!!reference method!!! for urea determination

> Clinical Significance

a. AZOTEMIA : elevated concentration of urea in blood
TYPES OF AZOTEMIA
PRERENAL RENAL POSTRENAL
- Due to reduced renal blood - Decreased renal function = - Due to obstruction of urine
flow compromised urea flow
- Less blood to kidneys, less excretion = elevated urea - Renal calculi, tumors of the
filtered in plasma bladder or prostate or
- Congestive heart failure - Acute or chronic renal severe infection
(CHF), shock, failure, glomerular
hemorrhage,and nephritis, acute tubular
dehydration necrosis

b. UREMIA / UREMIC SYNDROME: very high plasma urea concentration accompanied by renal
failure
* causes of decreased plasma urea concentration include low protein intake and severe liver disease
* differentation of elevated plasma urea concentration is aided by calculation of urea nitrogen:
creatinine ratio which is normally 10:1 to 20:1

B. CREATININE/ CREATINE
- Creatinine is formed from creatine and creatine phosphate in muscle and is excreted into the
plasma at a constant rate related to muscle mass
- plasma creatinine is inversely proportional to glomerular filtration rate (GFR)
- measurement is clinically use to determine sufficiency of kidney function and severity of kidney
damage, and to monitor progression of kidney disease

25
 - Reference value: Male = 0.9 – 1.3 mg/dL ( 80- 115 umol/L)
Female = 0.6 – 1.1 mg/dL (53 – 97 umol/L)
➢ METHODS
a. Chemical Method – Jaffe Reaction (end point)
- creatinine reacts with picric acid in alkaline solution to form a red-orange chromogen
- lacks specificity; ascorbic acid, blood-substitute products, cephalosporins, glucose, guanidine,
ketone bodies, protein, and pyruvate are the known Jaffe like chromogens that interferes with
the reaction
- Jaffe reagent : saturated picric acid and 10% NaOH
* Lloyd’s or Fuller Earth’s Method: use adsorbent to increase sensitivity and specificity
Lloyd’s reagent : sodium aluminum silicate
Fuller Earth’s reagent : aluminum magnesium silicate

b. Kinetic Jaffe method

-developed in order to increase specificity of the assay and for a more rapid creatinine
determination
- serum is mixed with alkaline picrate and the rate of change in absorbance is measured
-rapid, inexpensive, and easy to perform
- false increase levels due to interference with alpha keto acids and cephalosporins

c. Coupled enzymatic methods

- enzymes used include creatininase, creatinase, sarcosine oxidase and peroxidase
- adapted on dry slide analyzers
!measure ammonia colorimetrically or with ISE

d. IDMS : Reference method!!!

➢ Clinical Significance
Increased serum creatinine Decreased serum creatinine
Impaired renal function Decreased muscle mass
Chronic nephritis Advanced and severe liver disease
Congestive heart failure Pregnancy
Inadequate dietary protein

C. URIC ACID : product of purine catabolism (adenine and guanine)

- formed from xanthine by the action of xanthine oxidase in the liver and intestine
- relatively insoluble in plasma; can be deposited in joints and tissues if present in high levels
- majority in plasma exists as monosodium urate
- Reference intervals (uricase method) : Male = 3.5 – 7.2 mg/dl (0.21 – 0.43 mmol/L)
Female = 2.6 – 6.0 mg/dl (0.16 – 0.36 mmol/L)
Child = 2.0 – 5.5 mg/dL (0.12 – 0.33 mmol/L)

➢ METHODS
1. Oxidation-Reduction reaction : CARAWAY method
- based on the oxidation of uric acid in a protein-free filtrate, with subsequent reduction of
phosphotungstic acid in alkaline solution to tungsten blue
! subject to interference with turbidity and several common drugs
26
 2. Enzymatic method : URICASE method
- uricase catalyzes the oxidation of uric acid to allantoin
- measures the differential absorption of uric acid and allantoin at 293 nm; difference in
absorbance before and after incubation with uricase is proportional to the uric acid
concentration
- Coupled enzymatic methods measure the hydrogen peroxide produced as uric acid is
converted to allantoin; Peroxidase or catalase is used as chemical indicator of the reaction
! enzymatic UV method : needs special instrumentation
! enzymatic peroxidase( H2O2 production) : subject to interference with reducing subs
3. IDMS – Reference Method !!!

➢ Clinical significance
1. Hyperuricemia: GOUT - monosodium urate precipitates from super- saturated body fluids
- GOUTY ARTHRITIS: disease primarily diagnosed in men; imparts pain and inflammation in the
joints due to accumulation of monosodium urate
- plasma uric acid conc of >6.0 mg/dl
- tophi are crystalline deposits of urates in tissues seen in severe cases
> other causes of hyperuricemia
* increased nuclear metabolism : leukemia, lymhoma, MM, polycythemia vera, hemolytic and
megaloblastic anemias
* Chronic renal disease
* Lesch- Nyhan Syndrome : deficiency of hypoxanthine-guanine phosphoribosyl transferase
(HGPRT)
* !!! secondary to GSD!!!, ethanol consumption, toxemia of pregnancy and lactic acidosis

2. Hypouricemia: usually secondary to severe liver disease or defective tubular reabsorption

(Fanconi’s syndrome)

D. AMMONIA : formed in deamination of amino acids during protein metabolism; aided by normal flora
in the intestinal lumen
- removed from the circulation and converted to urea in the liver
- blood ammonia concentration provides useful information in hepatic failure, Reye’s syndrome,
and inherited deficiencies of urea cycle enzymes

➢ METHODS
- Blood sample for ammonia testing must be placed in ice immediately after collection

A. Method by Conway
- first analytic method for ammonia; based on the property of ammonia to volatilize to a
separate compound in a microdiffusion chamber
- Ammonia gas from the sample diffuses into a separate compartment and is absorbed in a
solution containing a pH indicator. The amount of ammonia was determined by titration

27
 Chemical methods
Ion-selective electrode Diffusion of NH3 through Good accuracy and precision,
selective membrane into membrane stability may be
NH4Cl causing pH change, problem
which is measured
potentiometrically
Spectrophotometric NH3 + bromphenol blue to
blue dye
Enzymatic methods
GLDH GLDH Most common on automated
NH4+ + 2-oxoglutarate + instruments; accurate and
NADPH + H+ to glutamate + precise
NADP + H2O

➢ Clinical Significance
- Free ammonia is toxic!
a. Reye’s Syndrome : most common in children; frequently preceded by a viral infection and
administration of aspirin

V. ENZYMES
- are specific biologic proteins that catalyze biochemical reactions without altering the
equilibrium point of the reaction or being consumed or changed in composition.The other
substances in the reaction are converted to products.
- present in all body tissue thus they are detectable in serum following cellular injury,
degradation of cells or from storage areas
- measured in terms of their activity
- they have the capacity to recognize and bind only one or few molecules and catalyze a single
reaction or a limited number of chemical reactions
- a Substrate is the substance to which the enzyme interacts with

➢ Enzyme structure
a. Active site: cavity where the substrate of the enzyme interacts with particular charged amino acid
residues
b. Allosteric site : a cavity other than the active site and may bind to regulatory molecules
c. Cofactor : nonprotein molecule necessary for enzyme activity
• Activators : Inorganic cofactors (chloride or magnesium ions)
• Coenzyme : Organic cofactors such as nicotinamide adenine dinucleotide
d. Prosthetic group : Coenzyme bound tightly to the enzyme
e. Holoenzyme : Apoenzyme (enzyme portion) + cofactor
f. Proenzyme/ Zymogen : inactive form of enzyme
* Isoenzyme : different forms of the same enzyme in an individual
* Isoform : results when the enzyme undergo posttranslational modification

28
➢ Enzyme Kinetics : Catalytic mechanism of enzymes
- General relationship among enzyme, subtrate and product
ENZYME + SUBSTRATE ENZYMESUBSTRATE ENZYME + PRODUCT

Factors influencing enzymatic reactions

Substrate concentration Reaction rate increases as more substrate is
added
Enzyme concentration Higher enzyme concentration mean faster rate
of reaction
Presence of inhibitors a. competitive inhibitor
- compete with the subtrate in binding to the
active site of the enzyme
- inhibiton reversed by adding more substrate
b. noncompetitive inhibitor
-binds to enzyme in places other than the active
site
- may be reversible or irreversible
c. uncompetitive inhibitor
- inhibitor binds to Enzyme Substrate complex
- adding more substrate will just result to more
inhibition
Temperature - Increase temp means increase in enzyme
activity
- Optimal temp for enzymes is usually close
to physiologic temp which is 37oC
- Denaturation of enzyme occurs when temp
starts to reach 40 – 50oC
pH - Most reactions occur at pH 7 to 8
Ionic strength (salt an protein concentration) - Too high ionic strength of the solution
causes a drop in enzyme activity

Enzymatic Reactions
1. Michaelis-Menten Hypothesis : a substrate readily binds to free enzyme at a low-substrate
concentration. With the amount of enzyme exceeding the amount of substrate, the reaction rate steadily
increases as more substrate is added
-Km: Michaelis-Menten constant -expression of the relationship between the velocity of an enzymatic
reaction and substrate concentration; indicates amount of substrate needed for a particular enzyme
reaction

𝑽𝒎𝒂𝒙 (𝑺)
V= V = velocity of reaction
𝑲𝒎+(𝑺)
Vmax = maximum velocity

a. First order kinetics : reaction depends only on enzyme concentration

b. Zero order kinetics : reaction is directly proportional to substrate concentration

2. Lineweaver- Burk transformation of MM hypothesis : a double-reciprocal plot of the Michaelis-Menten

constant which yields a straight line
29
 Bishop, Michael L., et.al. (2010). Clinical Chemistry – Principles, Procedures and Correlations. 6th ed. Lippincott Williams and Wilkins.

Competitive inhibition Uncompetitive Nonompetitive

inhibition inhibiton
Km Increase Reduced Unaffected
Vmax Unaffected Reduced reduced

➢ Methods to measure extent of enzyme reaction

a. Fixed time : the reactants are combined together and the reaction proceeds for a designated
time. The reaction is the stopped and the measurement is made

b. Continuos monitoring/ Kinetic assay : multiple measurements of change in absorbance are

made during the reaction; PREFERED than fixed-time method because linearity can be checked

Units for expressing Enzymatic Activity

• International Unit (IU or U) : 1 umol of substrate/ minute
• Katal Unit (KU) : 1 mole of substrate/ second

➢ Enzyme Classification
Class Function Example
1. Oxidoreductases For removal or addition of DEHYDROGENASES AND
electrons (redox reaction) OXIDASES

2. Transferases For transfer of a chemical KINASES, PHOSPHORYLASES

group other than hydrogen AND TRANSFERASES
from one substrate to another

3. Hydrolases For hydrolysis or splitting of a HYDROLASES AND

bond by the additon of water PHOSPHATASES
30
 4. Lyases For removal of groups from ALDOLASE AND
substrates without hydrolysis DECARBOXYLASES
-product will contain a double
bond
5. Isomerases For intramolecular Glucose phosphatase
arrangement of substrate isomerase
compound Ribose phosphate isomerase
6. Ligases For joining of two substrate Glutathione synthase
molecules

➢ MAJOR CLINICAL ENZYMES

A. LIVER ENZYMES
1. AMINOTRANSFERASES: Pyridoxal Phosphate serves as a coenzyme in amino transfer (Vit B6)
1.a. Aspartate Amino Transferase (AST) : serum glutamic-oxaloacetic transaminase (SGOT)
-involved in the transfer of an amino group between aspartate and α-keto acids
• Major source: Heart, Liver, skeletal muscle and kidneys
• Minor source: RBC and pancreas
Reference value : 5 to 30 U/L

>Assay for enzyme activity: KARMEN method

Aspartate + α-ketoglutarate AST oxaloacetate + glutamate

Oxaloacetate + NADH + H Malate + NAD

- uses Malate dehydrogenase as indicator and monitors change in absorbance at 340nm

- Hemolysis must be avoided because it causes a significant increase in serum AST concentration

Clinical Significance:
-mainly for evaluation of hepatocellular and skeletal muscle disorders
Pronounced Elevation Moderate elevation Slight elevation
Acute Hepatocellular disorders Cirrhosis Pulmonary infarction
Viral Hepatitis Skeletal muscle disorders Pericarditis
Myocardial Infarction Primary or metastatic Cerebrovascular accident
Acute pancreatitis Carcinoma of the liver
Muscular dystrophy
Congestive heart failure
Billiary obstruction
Infectious mononucleosis

1.b. Alanine amino transferase (ALT): serum glutamic-pyruvic transaminase (SGPT)

- catalyzes the transfer of an amino group from alanine to α-ketoglutarate with the formation of
glutamate and pyruvate
• Major source: Liver
• Minor source: kidney, pancreas, RBC, heart muscle, skeletal and lungs
Reference Value : 6-37 U/L

31
 >Assay for enzyme activity: Coupled enzymatic reaction
- uses LDH as indicator with subsequent measurment of loss of absorbance of NADH
- relatively unaffected by hemolysis
Alanine + α-ketoglutarate AST pyruvate + glutamate

LD
Pyruvate + NADH + H lactate + NAD

Clinical significance
-mainly elevated in heptocellular disorders; it is more liver specific than AST
-more sensitive and specific screening test for postransfusion hepatitis or after exposure to
infectious biological sample
• De Ritis ratio (ALT:AST) >1.0 seen in Acute hepatitis

2. Gamma Glutamyl Transferase (GGT) : involved in the transfer of the γ-glutamyl residue
from γ-glutamyl peptides to amino acids, H2O, and other small peptides.
- Glutathione serves as the major gamma glutamyl donor
• Major source: kidney, brain, prostate, pancreas, and liver (canaliculi of hepatic cells)
Reference Value: 6 – 45 U/L (Male) 5 – 30 U/L (Female)

>Assay for enzyme activity

- γ-glutamyl-ρ-nitroanilide is the most commonly used substrate
- gamma-glutamyl residue is transferred to glycylglycine, releasing p-nitroaniline, which has
a strong absorbance at 405 to 420 nm
-RBC has no GGT so hemolysis does not affect the results

Clinical significance
• Elevated in all hepatobilliary d/o with highest elevation in cases of hepatobilliary
obstruction
• Elevated levels may indicate chronic alcoholism
• Diabetes mellitus, acute pancreatitis and myocardial infarction

B. Phosphatases
a.Alkaline phosphatase (ALP) :catalyze the hydrolysis of various phosphomonoesters at an
alkaline pH; Zinc is a major component of the enzyme
-optimal pH for the reaction is at ph 9.0 to 10.0 and requires Mg as activator
• Major source : intestine, liver, bone(osteoblasts), spleen, placenta, and kidney
Reference Value : 30 to 90 U/L
Major Isoenzymes Carcinoplacental ALP
1. Liver ALP 1. Regan: found in lung, breast and ovarian cancers
2. Bone ALP 2.Nagao : found in adenocarcinoma of pancreas and bile
3. Placental ALP duct, and in pleural cancer
4. Intestinal ALP

>Methods

32
 1. Electrophoresis : Most Anodal – Liver, Bone, Placental, and Intestinal – Least Anodal
2. Heat Fractionation / Stability Test : Most Stable - *Regan (if present), Placental,
Intestinal, Liver, Bone – Least Stable

3. Chemical Inhibiton
Inhibited by:
• phenylalanine : all( including carcinoplacental) except liver and bone
• Levamisole : liver and bone ALP
• L- leucine : Nagao
• 3M urea : Placental and intestinal

4. Bowers and Mc Comb by Szasz : IFCC recommended method; most specific

Methods Substrate End Products

Bodansky β-glycerophosphate Inorganic phosphate + Glycerol
Shinowara, Jones and Reinhart
Bowers McComb ρ-nitrophenylphosphate (PNPP) ρ-nitrophenyl (Yellow)
Bessy, Lowry and Brock
King and Armstrong Phenylphosphate Phenol
Moss Alpha naphthol phosphate Alpha naphthol

Clinical Significance : most diagnostic for evaluation of hepatobilliary and bone disorders
Pronounced Elevation (>5x Moderate Elevation (3 -5x than Slight Elevation (up to 3x than
than normal) normal) normal)
Bile duct obstruction Metastatic bone tumors Viral heptitis
Paget’s disease Osteomalacia Pregnancy
Hyperparathyroidism Infectious Mononucleosis Growth (children)
Biliary cirrhosis

b. Acid Phosphatase (ACP):catalyze the same reaction as that of ALP, but the reaction
happens at pH 5
• Major Source: Prostate
• Minor source: RBC, platelets, liver and bone
Reference value : Prostatic ACP – 0 to 3.5 ng/ml

>Methods
* delay in assay : serum must be acidified at a pH lower than 6.5 or frozen
*Prostatic ACP is inhibited by L-tartrate, but not the red cell ACP
* affected by hemolysis so serum sample must be free of hemolysis
Method Substrate End product
Gutman and Gutman Phenyl phosphate Inorganic phosphate
Shinowara PNPP ρ-nitrophenol
Babson, Reed and Philips Alpha naphthyl phosphate Alpha naphthol
Roy and Hillman Thymolphthalein Free thymolphthalein
monophosphate
33
 Clinical Significance
-increased in prostatic carcinoma, bone diseases, Paget’s disease, breast cancer with
bone metastases and in Gaucher’s disease.
-in investigation of rape cases; seminal fluid ACP in vaginal washings may stay elevated
for up to 4 days

C. Muscle enzymes
A. Creatine Kinase (CK) : catalyze reversible phosphorylation of creatine by ATP
- a dimeric molecule composed of two different monomers- M and B

CK
Creatine + ATP Creatine phosphate + ADP

• Major source : skeletal muscle, heart and brain

Reference Value : Male = 15 – 160 U/L
Female = 15 – 130 U/L
CK-MB = <6% of total CK

>Isoenzymes
CK - 1 CK – BB (BRAIN TYPE) MOST ANODAL AND MOST
LABILE
CK - 2 CK – MB (HYBRID TYPE) 20% IN CARDIAC TISSUE
CK - 3 CK-MM (MUSCLE TYPE) LEAST ANODAL
*MAJOR ISOENZYME IN SERUM
Macro CK CK-BB complexed with Migrates between CK-MM and
Immunoglobulin CK-MB
Mitochondrial CK ( CK - Mᵢ) Not present in normal serum Migrates cathodal to CK-MM

>Methods:
Forward/ direct reaction Tanzer – Gilvarg Assay Coupled with PK – LD – NADH system

CK
Creatine + ATP Creatine PO4+ADP

PK
ADP + phosphoenolpyruvate pyruvate +
ATP

Pyruvate + NADH + H+LD lactate + NAD+

Reverse / indirect reaction Oliver – Rosalki Coupled with Hexokinase – G6PD – NADP
*most commonly used
CK
method Creatine PO4 + ADP Creatine + ADP

KH
ATP + glucose ADP + glucose-6-PO4

34
 Glucose-6-PO4 + NADPH+G-6-PD 6-
phosphogluconate + NADPH

*Adenylate Kinase(AK) is an enzyme that is released after hemolysis; it can interfer with CK assays
Clinical significance
-primarily increased in Duchenne’s muscular dystrophy and MI
-Others include pulmonary infarction, Reye’s syndrome, strenous exercise
Hypothyroidism, polymyositis and intramascular injection

b. Lactate Dehydrogenase (LD) : catalyzes the interconversion of lactic and pyruvic acid
- NAD serves asits coenzyme
- the most widely distributed enzyme - nonspecific
- a tetrameric molecule with four subunits of two possible forms-H and M
• Major source : RBC, platelets, heart, kidney and liver
Reference Value : 100 – 255 U/L

>Isoenzymes and their significance

* LD flipped pattern : LD 1 > LD 2 – suggestive of acute myocardial infarction

Isoenzyme Tissue Disorder

LD -1 (HHHH) AMI and Hemolytic anemia
- Most anodal
LD – 2 (HHHM) Heart and RBCs Megaloblastic anemia, acute
- Major form in the serum renal infarct and hemolytic
anemia
LD – 3 (HHMM) Lung, lymphocyte, spleen and Pulmonary embolism, extensive
pancreas pulmonary pneumonia,
lymphocytosis, acute
pancreatitis and carcinoma
LD – 4 (HMMM) Liver Hepatic injury or inflammation
- Cold labile
LD – 5 (MMMM) Skeletal muscle Skeletal muscle injury
- Least anodal
- Cold labile
LD – 6 Arteriosclerotic cardiovascular
Alcohol dehydrogenase failure

>Assay for enzyme activity

* source of error : Hemolyzed sample because RBC has 100 to 150 times LD
Wacker Method – Forward/ direct reaction - Lactate to pyruvate
- Most commonly used method
Wrobleuski La Due – Reverse/ indirect reaction - Pyruvate to lactate
- Much faster than forward reaction

>Clinical Significance
Pronounced Elevation Moderate Elevation Slight Elevation
Pernicious anemia Leukemia Viral hepatitis
35
 Hemolytic anemia Muscular dystrophy Cirrhosis
Hepatitis Other malignancies AMI
Renal infarction Pulmonary infarction
Nephrotic syndrome

D. Pancreatic Enzymes
1. Amylase (AMS) : catalyze the breakdown of starch and glycogen
-two types: S type (salivary) and P type (pancreatic)
• Major source: Salivary gland and pancreas
Reference Value: Serum= 25–130 U/L
Urine=1–15 U/h

>Assay for enzyme activity

* Starch : General substrate
Amyloclastic Measures diasappearance of starch
Saccharogenic Measures appearance of the product
- Reference method ; expressed in Somogyi
units
Chromogenic Measures the increasing color from production of
product coupled with a chromogenic dye
Continuous monitoring Coupling of several enzyme systems to monitor
amylase activity

>Clinical Significance
1. Acute pancreatitis : highly elevated; major diagnostic significance
2. Salivary gland lesions : Parotitis and mumps
3. Perforted peptic ulcers
4. Morphine administration
5. Pancreatic carcinoma and pseudocyst

2.Lipase(LPS) : hydrolyzes the ester linkages of fats to produce alcohols and fatty acids
- catalyzes the partial hydrolysis of dietary triglycerides in the intestine
- most specific pancreatic marker
• Major source: pancreas
• Minor source: Stomach and Small intestine
Reference Value: 0 – 1.0 U/ml

>Assay for Enzyme Activity

Cherry Crandal - Reference method
- Uses olive oil as subtrate and measures
liberated fatty acids by titration after 24 hr
incubation

Clinical Significance
-mainly for diagnosis of Acute pancreatitis; more specific to pancreatic d/o than AMS
-useful in differentiating serum Amylase elevation as a result of pancreatic versus salivary
involvement

36
 PANCREATIC PROFILE
Start of elevation Peak hour Duration (Days)
Amylase 2 – 12 hr 24 hr 3 – 5 days
Lipase 2 – 4 days 5 days to 2 weeks

➢ Other Clinically significant Enzymes

1. 5’ Nucleotidase (5’N) : acts only on nucleoside-5'-phosphates, such as adenosine-5'-monophosphate
(AMP), releasing inorganic phosphate
- Marker for hepatobilliary diseases
Reference Valuei: 0 – 1.6 units

2. Glucose – 6 – phosphate dehydrogenase (G6PD) : catalyzes the oxidation of glucose-6phosphate to 6-

phosphogluconate or the corresponding lactone
- maintains NADPH in the reduced form in erythrocytes
- deficiency of this enzyme results to drug- induced hemolytic anemia, particularly after taking
primaquine (anti-malarial drug)
- Red cell hemolysate is used to assay enzyme deficiency; Serum is for evaluation of enzyme elevation

3. Cholinesterase
- Indicator of possible pesticide poisoning: DECREASED enzyme activity

a. Acetylcholinesterase : true cholinesterase

-found in erythrocytes, the lungs and spleen, nerve endings, and the gray matter of the brain
-for hydrolysis of acetylcholine released from nerve endings to generate nerve impulses

b.Acylcholine acylhydrolase : pseudocholinesterase/ serum cholinesterase

-found in the liver, pancreas, heart, white matter of the brain, and serum
-unknown biological role

4. Drug metabolizing enzymes : transform xenobiotics into inactive, water-soluble compounds for excretion
through the kidneys
-cytochrome P450 (CYP450) most common enzyme in this class

VI. KIDNEY FUNCTION TESTS

A. Tests for Glomerular Filtration Rate
>Clearance: renal clearance relates to the urinary excretion of material to the plasma concentration of
the same material
- Plasma concentration is inversely proportional to clearance

𝑼𝑽
Cx = C = clearance in ml/min P = con of analyte in plasma
𝑷
U = conc of analyte in urine
V =vol of urine in ml/24 hr

1. Inulin Clearance: Reference method but not routinely done

37
 - requires continuous IV infusion and timed urine collections
- exogenous
Reference Values: Male = 127 ml/min
Female = 118 ml/min

2. Creatinine Clearance: most widely used marker for GFR

- endogenous, fair rate of production and freely filtered by the glomerulus
- measure of the amount of creatinine eliminated from the blood by the kidneys
- the abbreviated Modification of Diet in Renal Disease (MDRD) equation
includes four variables—serum (plasma) creatinine concentration, age, gender
(sex), and ethnicity

Reference Values : Male = 85 – 125 ml/min

Female = 75 – 112 ml/min
Increase Creatinine Clearance Decreased Creatinine Clearance
High cardiac output, pregnancy, burns and Impaired kidney function, shock, dehydration,
carbon monoxide poisoning hemorrhage, and CHF

3. Urea clearance, Cystatin C and Beta trace proteins

VII. Liver Function Tests

-80% destruction in the liver before results become abnormal

>Functions of the Liver

1. Synthetic: production and secretion of plasma proteins, carbohydrates, lipids, LPP, clotting
factors, ketone bodies and enzymes
2. Conjugation of Bilirubin
3. Detoxification and Drug metabolism
4. Excretory and Secretory : excretion of bile
5. Storage : glycogen, fat soluble and water soluble vitamins

A. Tests for Synthetic Function : generally methods used to measure proteins

B. Tests for measuring Conjugation and Excretory Function:

BILIRUBIN : major metabolite of HEME; principal pigment in Bile

Reference Values: Conjugated bilirubin = 0 – 0.2 mg/dl (0 – 3 umol/L)
Unconjugated bilirubin = 0.2 – 0.8 mg/dl ( 3 – 14 umol/L)
Total bilirubin = 0.2 – 1.0 mg/dl (3 – 17 umol/L)

Unconjugated Bilirubin Conjugated Bilirubin

- Bilirubin 1; Hemobilirubin - Bilirubin 2; Cholebilirubin
- Water insoluble - Water soluble
- Indirect reacting - Direct reacting
- Prehepatic bilirubin - Posthepatic bilirubin

38
*Delta bilirubin : conjugated bilirubin bound to albumin; formed due to prolonged elevation of conjugated
bilirubin in cases of billiary obstruction

>Bilirubin Assay
- works on the principle of diazotization of bilirubin to produce azobilirubin
- Unconjugated bilirubin reacts slowly, so an accelerator is necessary for it to react
- removing the accelerants from the reaction will allow measurement of Conjugated bilirubin
which is the direct reacting bilirubin

>Specimen Collection and Storage

- specimen should be protected from light; if left unprotected from light, it may reduce by
30% - 50 % per hour

Evelyn – Malloy Accelerator : Methanol

End product : pink to purple azobilirubin
Jendrassik - Grof Accelerator : Caffeine sodium benzoate
End product : pink to blue azobilirubin
- More sensitive and most commonly used
*Total Bilirubin is measured 15 minutes after the additon of the accelerator
Direct bilirubin ( first step) + Indirect bilirubin (after add’n of accelerator) = Total bilirubin

Clinical Significance : JAUNDICE – from French word jaune, which means yellow
- yellow discoloration of the skin, eyes, and mucous membranes
-also known as icterus and hyperbilirubinemia
-results when bilirubin levels in blood exceeds 2 mg/dl

>Classification
A. Pre-hepatic jaundice: due to HEMOLYTIC d/o
-increased levels of bilirubin being presented to the liver; the liver in term functions to
its maximum capacity
-rarely exceeds 5mg/dl
B. Hepatic jaundice
Gilbert Syndrome - Defect in bilirubin transport
- Decreased hepatocellular uptake of bilirubin
Crigler – Najjar - Defect in conjugation
➢ Type I : deficiency in UDPGT
(uridine diphosphoglucuronyl transferase)
-total absence of B2 and prone to
Kernicterus

➢ Type II : partial deficiency only

Dubin – Johnson syndrome - Blocked excretion of bilirubin in the
canaliculi

39
 - Defect in ATP-binding cassette (ABC)
canalicular multispecific organ anion
transporter – MDR2/cMOAT
- Liver biopsy shows dark pigmentation
Rotor Syndrome - Clinically similar to Dubin – Johnson but no
dark pimentation in liver biopsy
Lucey – Driscoll Syndrome - There is circulating inhibitor/ antibody to
UDPGT so no conjugation

C. Post – hepatic Jaundice : impaired bilirubin excretion due to bile duct obstruction

Changes in concentration of Bilirubin in different types of disorders causing Jaundice

SERUM
Type of Jaundice Total Bilirubin Conjugated Bilirubin Unconjugated Bilirubin
Prehepatic Increased Normal Increased
Hepatic
Gilbert disease Increased Normal Increased
Crigler-Najjar Increased Decreased Increased
syndrome
Dubin-Johnson Increased Increased Normal
Rotor syndrome Increased Increased Normal
Jaundice of the Increased Normal Increased
newborn
Posthepatic Increased Increased Increased

URINE
Bilirubin Urobilinogen
Pre-hepatic Not present Increased
Hepatic Positiive Increased
Post-hepatic Positive Normal to increased

VIII. CARDIAC PROFILE

>MYOCARDIAL INFARCTION : myocardial necrosis due to prolonged ischemia

Cardiac Markers and their time profiles

Onset Peak hour Duration
CK - MB 4 – 6 hr 12 – 24 hr 2 – 3 days
AST 6 – 8 hr 24 hr 5 days
LD 12 – 24 hr 72 hr 10 days
Myoglobin 1 – 4 hr 6 – 9 hr 18 – 24 hr
Troponins 4 – 10 hr 12 – 48 hr 4 – 10 days
*the sustained elevation of Troponins make them a more definitive marker for AMI. Moreover, unlike CK – MB,
Troponins are not present in normal sera

IX. ELECTROLYTES
-Ions that are either negatively charged (ANION) or positively charged (CATION)
40
 Significance
Volume and osmotic regulation Sodium, Potassium and Chloride
Myocardial rythym and contractility Potassium, Magnesium and Calcium
Neuromuscular excitability
Cofactors Magnesium, Calcium and Zinc
Regulation of ATP pumps Zinc
Acid-Base balance Bicarbonate, Potassium and Chloride
Production and use of ATP from glucose Magnesium and Phosphate
- Water in 40% to 75% of total body weight
-Active transport mechanism that requires energy to move ions across cellular membranes
- Diffusion passive movement of ions across cellular membranes
- Osmolality physical property of a solution based on conc of solutes per kilogram of solvent;
Normal plasma osmolality: 295 mmol/L

> RENIN – ANGIOTENSIN – ALDOSTERONE SYSTEM (RAAS)

- maintains blood volume and ensure good perfusion of blood to tissues

ANGIOTENSIN
ANGIOTENS ANGIOTENS • VASOCONSTRICTION

INOGEN RENIN ANGIOTENSIN I CONVERTING

IN II
• SECRETION OF
ALDOSTERONE
ENZYME (ACE)

A. SODIUM (Na)
- Most abundant cation in ECF
- plasma conc depends on intake and excretion of water, and to renal regulation but to a
lesser degree
Reference Value : 135 – 145 mmol/L

>Methods of Determination:
1. Atomic absorption spectrometry (AAS)
2. Flame Emission Spectrometry (FES) :Yellow flame
3. Ion Selective Electrode (ISE) : Glass aluminum silicate ; Method of choice

>Clinical Significance

Hypernatremia Hyponatremia
Reduced water intake Increased water retention
- Defect in thirst center - advanced renal failure
- nephrotic syndrome
- hepatic cirrhosis
- CHF

Increased water loss Increased sodium loss

- Gastrointestinal : vomiting, osmotic diarrhea - Hypoadrenalism
- Cutaneous loss: sweating and fever - Salt-losing nephropathy

41
 - Respiratory loss: hyperventilation and fever - Diuretics use
- Renal loss: DI and osmotic diuresis - Prolonged vomiting and diarrhea; burns
Increased sodium intake Excess water intake
Increased retention Water imbalance
- hyperaldosteronism -SIADH

• Pseudohyponatremia : spurious reduction in serum sodium concentration because of a systematic error

in measurement
- most common cause : in vitro hemolysis
B. POTASSIUM: major intracellular cation; counterbalance of sodium
- 20 times greater inside the cell than outside
Reference Value: Serum = 3.5 – 5.1 mmol/L

>Methods of determination:
* Hemolysis must be avoided because the RBCs have a high content of potassium
*Heparin is the anticoagulant of choice
1. ISE :valinomycin membrane ; method of choice
2. FES : Purple flame
3. AAS and Spectrophotometry

>Clinical Significance
Hyperkalemia Hypokalemia
Extracellular shift Intracellular shift
- acute acidosis - alkalosis
- hemolysis - insulin overdose
- vigorous exercise - nutritional recovery
- chemotherapy and leukemia - barium poisoning
Decreased renal excretion GI loss
- hypoaldosteronism - vomiting, diarrhea, and laxative abuse
- acute or chronic renal failure - malabsorption
- addison’s dse - intestinal tumor
- diuretics use - chemotherapy
Artifactual Renal loss
- sample hemolysis - primary aldosteronism
- thrombocytosis - diuretics use; Renal tubular acidosis
- prolonged tourniquet application - acute leukemia
- excessive clenching of fist - hypomagnesemia

• Pseudohyperkalemia:Thrombocytosis, severe leukocytosis and in vitro hemolysis

C. CHLORIDE : major extracellular anion; counterion of Sodium

Reference Value : 98 – 107 mmol/L
- chloride shift : maintenance of electroneutrality

42
 https://medical-dictionary.thefreedictionary.com/chloride+shift

>Methods of Determination
* Lithium Heparin is the anticoagulant of choice
* Sweat can be used for measurement
* Bromide, cyanide and cysteine interferes with the measurement
1. Amperometric – Coulometric Titration (Cotlove Chloridemeter)
coulometric generation of silver ions, which combine with Cl to quantitate the Cl
concentration
2. Mercurimetric Titration By Schales & Schales : end product HgCl2(blue violet)
3. ISE : most commonly used

>Clinical Significance
Hyperchloremia Hypochloremia
Renal tubular acidosis and Metabolic acidosis Prolonged vomiting
Prolonged diarrhea Hypoaldosteronism
DI and Salicylate intoxication Metabolic alkalosis
Primary hyperparathyroidism Salt losing nephropathy

>Cystic Fibrosis: inherited d/o of exocrine glands; production of thick mucus secretions, elevated sweat
electrolytes, overactivity of autonomic nervous system and increased enzymatic constituents of saliva
• Diagnostic Test: Sweat Test ( increases Na and Cl)
• Pilocarpine is the most commonly used sweat inducer

D. BICARBONATE : second most abundant anion in the ECF

Total CO2 : Bicarbonate ion, Carbonic Acid and dissolved carbon dioxide
-major component of buffering system in blood; Acid-base imbalances causes an
imbalance in bicarbonate and carbon dioxide

> Specimen consideration

- Blood is collected anaerobically
- serum or Lithium heparin is suitable for analysis

E. Magnesium: fourth most abundant cation in the body and is the second most abundant

43
 intracellular ion
-53% in bones. 46% in muscle and other organs, and 1% in serum
Reference Value: 1.2 – 2.1 mEq/L

>Forms:
a. Free magnesium / Ionized form : physiologically active – 55%
b. Protein- bound : 30%
c. complexed with ions: 15%

>Factors for regulation of Magnesium level in blood

1. Parathyroid hormone (PTH) : increased renal and intestinal reabsorption of Magnesium
2. Aldosterone and Thyroxine: increased renal excretion of Magnesium

>Methods of Determination
1. Colorimetric Method : Calmagite, Formazen and Magnesium Thymol Blue
2. AAS : reference method
3. Dye-lake method: Titan Yellow

>Clinical Significance
Hypermagnesemia Hypomagnesemia
- Decreased excretion Decreased absorption
- acute or chronic renal failure - malabsorption
- increased intake - diarrhea, vomiting and laxative use
- Dehydration - pancreatitis
- Hypothyroidism Increased Renal Excretion
- Hypoaldosteronism - tubular d/o
- Hypopituitarism - glomerulonephritis
- Bone carcinoma - pyelonephritis
- Bone metastases Increased Endocrine Excretion
- Hyperparathyroidism
- Hyperaldosteronism
- Hyperthyroidism
- Hypercalcemia
- Diabetic ketoacidosis

F. CALCIUM
- absorbed in the duodenum at an acidic pH
- 99% is found in bone and 1% in plasma; exist as Ionized (50%), protein bound (40%) and
complexed with anions (10%)

Reference Values: Total Calcium (serum or plasma)

- Child <12 y/o = 2.20–2.70 mmol/L (8.8–10.8 mg/dL)
- Adult = 2.15–2.50 mmol/L (8.6–10.0 mg/dL)
Ionized Calcium (serum)
- Child = 1.20–1.38 mmol/L (4.8–5.5 mg/dL)
- Adult = 1.20–1.38 mmol/L (4.8–5.5 mg/dL)

44
 >Regulation of Calcium levels in blood
1. Parathyroid Hormone (PTH) : stimulated when calcium levels decrease
activates bone resorption, stimulates tubular reabsorption and production of Vitamin D
2. Vitamin D3 (cholecalciferol) : comes from diet and from exposure to sunlight
- enhance action of PTH on bone resorption and for effective absorption in intestines
3. Calcitonin : acts when conc of Calcium in blood increases; inibits action of PTH and Vit D

>Methods of Determination
- specimen must be collected anaerobically and sample must be capped at all times
1. Dye-binding reactions : ortho-cresolphthalein complexone (CPC) and arsenzo III dye
2. Precipitation and Redox Titration : Clark Collip precipitation and Ferro Ham Chloroanilic Acid
3. EDTA titration method, FES, ISE and AAS (Reference method)

> Clinical Significance

Hypercalcemia Hypocalcemia
Primary hyperparathyroidism Primary hypoparathyroidism
Hyperthyroidism Magnesium imbalances
Malignancy and MM Hypoalbuminemia
Increases Vitamin D Vitamin D deficiency
Prolonged immobilization Renal disease

I. PHOSPHORUS: 80% - 85% is present in bones and 15% is present as inorganic PO4 in the extracellular
fluid
- Important constituent of RNA and DNA (as phosphate)
- Entirely distributed in the tissues

> Phosphorus Homeostasis

1. PTH: increase renal excretion therefore lowering its blood concentration
2. Vitamin D: increase absorption in intestine and renal reabsorption
3. Growth Hormone: reduce renal excretion of phosphates

Reference Values: Neonates = 1.45 – 2.91 mmol/L

Child (15 and below) = 1.07 – 1.74 mmol/L
Adult = 0.78 – 1.42 mmol/L

>Determination of Inorganic Phosphorus

1. Fiske Subbarow Method: Ammonium Molybdate method
- Commonly used and uses pictol as reducing agent
- Ammonium Molybdate-Phosphate complex is a colorless complex measured
by UV at 340nm

>Clinical Significance:
Hyperphosphatemia Hypophosphatemia
Acute or chronic renal failure Diabetic ketoacidosis
Increased intake from cow’s milk or laxatives Chronic obstructive pulmonary disease and
asthma
45
 Increased breakdown of cells Malignancy
Severe infections, extensive exercise, neoplastic Inflammatory bowel disease, anorexia nervosa
disorders and intravascular hemolysis and alcoholism

➢ Notes to remember!
- Anion Gap: difference between the unmeasured cations (Na and K) and unmeasured anions (Cl and HCO3)
-for monitoring of recovery form diabetic ketoacidosis
Reference value:
AG = Na – (Cl + HCO3) 8 – 16 mmol/L
AG = (Na + K) – (Cl + HCO3) 10 – 20 mmol/L
>increased in uremia or renal failure, ketoacidosis, poisoning (methanol, ethanol, ethylene glycol, and
salicylates), lactic acidosis, hypernatremia and INSTRUMENT ERROR
>decreased in hypoalbuminemia, hypercalcemia, hyperlipidemia and myeloma patients

X. BLOOD GASES AND pH MEASUREMENT

- for clinical management of respiratory and metabolic disorders, for detection of acid-base
imbalance and in monitoring therapy
>Definition of Terms
a. acid : subs than can yield hydrogen or hydronium ion when dissolve in water
b. base: yield hydroxyl ions
c. buffer: combination of weak acid or weak base and its salt; system that resist changes
in pH
d. pK: negative log of the ionization constant; is also the pH in which the protonated and
unprotonated forms are present in equal concentrations

>Buffer Systems: Regulation of Hydrogen ions

1. Bicarbonate – Carbonic acid system: major extracellular blood buffer
Importance : 1. H2CO3 dissociates into CO2 and H2O, allowing CO2 to be eliminated by
the lungs and H+ as water
2. changes in CO2 modify the ventilation (respiratory) rate
3. HCO3 concentration can be altered by the kidneys
2. Phosphate buffer system: exchange of sodium ion in the urine hydrogen ion filtrate
3. Hemoglobin and Plasma Proteins

>Organs for Regulation of Acid-base Balance

A. Lungs: control of CO2 excretion; rapid and sensitive in adjusting blood pH but short term only
B. Kidneys: reclaims HCO3 from the glomerular filtrate by excretion of acids; slow in response
but compensation is complete and long term

>Assessment of Acid-Base Homeostasis

HENDERSON – HASSELBACH EQUATION: expression of acid-base relationship
pKa = 6.1
𝒄𝑨−
pH = pKa = log A = base HA = weak acid
𝒄𝑯𝑨

>Parameters in Assessment of Acid-Base Balance

46
 pH measurement Normal pH : 7. 35 – 7.45
Acidosis : <7.35 Alkalosis: >7.45
Evaluation of ventilation Normal PCO2: 35 – 45 mmHg
Respiratory alkalosis: <35 mmHg
Respiratory acidosis: >45 mmHg
Evaluation of metabolic process Normal HCO3: 21 – 28 mEq/L
Metabolic acidosis: <21 mEq/L
Metabolic alkalosis: > 28 mEq/L
Degree of Oxygenation Normal PO2: 80 – 100 mmHg

>ACID-BASE DISORDERS
Compensation Causes
Metabolic Acidosis Reduction in Hyperventilation Renal acidosis
bicarbonate content of - Increased rate or - Uremic acidosis
the body depth of - Renal tubular
respiration acidosis
Extrarenal acidosis
- GI loss of
bicarbonate
- Ingestion of acids
Organic acidosis
- Lactic acidosis
- ketoacidosis

Metabolic Alkalosis Increase in serum Hypoventilation GI loss of bicarbonate

bicarbonate - decreased - vomiting
breathing rate - gastric suction
hypokalemia
*least effective Increased renal
compensation excretion of acid
- diuretics use
- hyperaldosteronism
Respiratory Acidosis Accumulation of CO2 Tissue buffering of CO2 Lung disease
Increased renal - COPD
excretion of acid - Acute asthma
Hypokalemia
Barbiturate intoxication
Respiratory Alkalosis Excess CO2 loss Decreased renal Lung disease
reabsorption of HCO3 - Pneumonia
- Pulmonary
*has the most effective embolism
compensation - Pulmonary
congestion
Hypoxemia
Liver disease
Gram-negative sepsis

47
 Drugs : salicylates

>Mixed Acid-Base Disorders

- a clinical condition in which two or more primary acid-base disorders co-exist.
- a result of inappropriate compensation: excessive or insufficient
- blood pH will be determined by the dominant disorder

>Specimen Collection and Analysis

- Arterial blood ( Heparinized) is the specimen of choice; 0.05ml heparin/ ml of blood
- the form and concentration of heparin used in collection is the most coomon error encountered in specimen
collection
- most common error in analyzers is an error in operating temperature

>Specimen considerations
Direction of Change
Affected Parameter pH PCO2 PO2
Cause
1. Delay in analysis
-With tube stopper on Decreased Increased Decreased
-uncapped sample Increased Decreased Increased

2. Increased
Temperature Increased Decreased Decreased

3. Leukocytosis Decreased

4. Fever (for each Increase by 3% Decrease by 7%

degree)

>Methodologies:
I. Gasometer : Van Slyke
II. Electrodes : used in analyzers
a. pH : potentiometry; Glass electrode is the most commonly used
b. pO2: polarography – amperometry; Clarke electrode
c. pCO2 : potentiometry; Severinghaus electrode

XI. TRACE ELEMENTS AND VITAMINS

A. TRACE ELEMENTS: are metals, except selenium, the halogens, fluoride and iodine
-only <1 ug/g of wet tissue and ,0.01% of dry body weight

Element Function Effects of deficiency Effects of toxicity

Chromium Insulin action; glucose Impaired glucose Oxidative damage, skin
and lipid metabolism tolerance, insulin ulcers, contact
resistance and dermatitis, lung cancer
peripheral neuropathy

48
 Cobalt Hemoglobin synthesis, Symptoms due to lack Cardiomyopathy,
and component of Vit of Vit B12; anemia, goiter, heart failure,
B12 anorexia and growth hypothyroidism,
depression vomiting and diarrhea
Copper Cellular respiration, Menke’s kinky hair Wilson’s disease :
and collagen synthesis syndrome kayser- fleischer rings
Fluorine Prevents tooth decay Increased dental Mottled enamel
carries
Iodine Component of thyroid Goiter, Goiter and
hormone hypothyroidism, and thyrotoxicosis
cretinism in infants
Manganese Bone and connective Skeletal and muscle Least toxic; psychiatric
tissue defects disorders
Molybdenum DNA metabolism and Growth depression Anemia, goiter,
uric acid production thyrotoxicosis

Selenium Protects from oxidative

damage of lipid, gene
expression
Zinc Protein synthesis Low height in children, Nausea, vomiting and
acrodermatitis,and gastrointestinal
immune dificits irritation

B. VITAMINS : organic molecules required in small amounts for health, growth and
reproduction.
- completely depends on dietary intake (except Vit D)

Water – soluble Functions Deficiency Syndrome Toxicity

vitamin
Ascorbate ( Vit C) Redox Reactions and Scurvy Cramps, kidney stones,
Hydroxylation of diarrhea, and nausea
collagen
Biotin Cofactor in Dermatitis, glossitis, None
carboxylation rxn hair loss and anorexia
Cobalamin (Vit B12) Folate metabolism and Megaloblastic anemia None
DNA synthesis
Folate Transfer and use of one Megaloblastic anemia none
carbon units in DNA and neural tube
and amino acid defects
synthesis
Niacin ( nicotinic acid) Redox reactions; part Pellagra: Dermatitis, Flushing; hepatotoxic
of NAD and NADP Dementia and diarrhea
Thiamine (Vit B1) Coenzyme in Dry and wet beriberi, Headache, muscle
decarboxylation Wernicke – Kosakoff weakness, cardiac
syndrome arrythmia
Pantothenic Acid ( Vit Incorporated in Very high doses:
B3) coenzyme A Diarrhea

49
 Pyridoxine (Vit B6) Coenzymes in many Cheilosis, glossitis, Ataxia and sensory
reactions dermatitis and neuropathy
convulsions
Riboflavin (Vit B12) Cofactor Angular stomatitis and None reported
corneal vascularization
Vitamin A (Retinol) Vision in dim light; Squamous metaplasia, Drowsiness,
growth and night blindness headache,vomiting,
reproduction stupor and skin pealing
Vitamin D Absorption of calcium Rickets in children; Hypercalcemia, bone
and phosphorus; osteomalacia in adults; demineralization,
mineralization of bones tetany constipation and
and teeth muscle weakness
Vitamin E (tocopherol) Antioxidant, RBC Ataxia and Mild GI distress,
integrity and cellular spinocerebellar coagulopathies
respiration degeneration
Vitamin K Cofactor of Defective clotting and Excess may decrease
(phytomenadione) procoagulants bleeding d/o clotting time

PART III : ENDOCRINOLOGY

• Endocrine System: finely integrated system whereby the hypothalamus, the pituitary,
and target glands continually communicate through feedback inhibition and stimulation
to control all aspects of metabolism, growth and reproduction
• Anterior Pituitary gland (adenohypophysis) : secretes Growth Hormone(GH), Prolactin
(PRL), TSH, ACTH, FSH and LH
• Posterior Pituitary gland (neurohypophysis): storage and release of Oxytocin and
Vassopressin/ ADH.

>Hypothalamic – Hypophysisal Unit : Feedback Mechanisms

• Positive feedback system: an increase in the product results to elevation of the activity
of the system and the production rate

• Negative feedback system: an increased product results to dereased activity of the

system and the production rate

Bishop, Michael L., et.al. (2010). Clinical Chemistry – Principles, Procedures and Correlations. 6th ed. Lippincott Williams and Wilkins.

>Hormone Actions:
• Endocrine: secreted in one location and released into the blood circulation
• Paracrine: secreted in endocrine cells and released into interstitial space; binds to
specific receptor of adjacent cell and affects its functions

50
 • Autocrine: binds to specific receptor on cell of origin resulting to self-regulation of its
functions
>Classification of Hormones
Peptides and Glycoproteins Polypeptides Steroids Amines
Proteins
- Synthesized FSH, HCG, TsH ACTH, ADH, GH, - Cholesterol as Epinephrine,
and stored and Angiotensin, common norepinephrine,
w/in the cell Erythropoetin. calcitonin, gastrin, precursor T3 and T4
in the form of glucagon, insulin, - Aldosterone,
secretory oxytocin, PTH, cortisol,
granules MSH, PRL and estradiol,
- Hypothalamic Somatostatin progesterone,
hormones testosterone
and activated
Vit D3

>Hormones Secreted by the Anterior Pituitary Gland

A. GROWTH HORMONE (GH)/ Somatotropin: most abundant hormone produced by anterior PG

- structurally related to prolactin and human placental lactogen
- secreted in a pulsatile fashion; peaks during puberty and declines with increasing age
- has direct influence on anabolic (enhanced protein synthesis) and catabolic processes
- stimulated by exercise, physical and emotional stress, hypoglycemia, and by sex
hormones
• Reference Value: <7 ng/ml

>Clinical Significance
1. Acromegaly: overproduction of GH; mainly a result of pituitary tumor
- features include progressive enlargement of the hands and feet as well as growth of
facial bones, including the mandible and bones of the skull
*Gigantism : if GH overproducing tumor occurs before closure of the epiphyseal plate

2. GH Deficiency:
- Children: familial or due to tumors; results to depression of normal growth (dwarfism)
-Adult: due to structural or functionalabnormalities of pituitary gland

>Diagnostic Testing:
- a single random measurement of GH is not diagnostic
- specimen must be a fasting serum sample

GH deficiency Acromegaly
Screening Test Confirmatory Test Screening Test Confirmatory Test
Exercise Test: GH Insulin Tolerance test Measurement of Glucose Suppression
must be elevated : GOLD STANDARD Somatomedin C or Test
- If failure to - 24 hr or Insulin Like Growth - Using 75g glu
elevate, it need overnight factor 1 (IGF 1) load
confirmation minitoring of GH Normal response:
suppression of GH
51
 - Failure to rise in - IGF 1 is elevated
conc is in increased GH Failure to decline is
confirmatory of conc diagnostic of
GH deficiency acromegaly

B. PROLACTIN (PRL) : responsible for initiation and maintenance of lactation

- secretion is normally kept at low levels by the action of Dopamine
- considered as stress hormone and has vital functions in reproduction
- it is a unique hormone because its mode of regulation is tonic inhibition
- regulation is either negative feedback or positive feedback (lactation, more suckling
means more prolactin stimulation)
• Reference Value: Male = 1 -20 ng/ ml Female = 1 – 25 ng/ml
>Clinical Significance:
1. Hyperprolactinemia: most common endocrine abnormality encountered in the lab
* Prolactinoma: PRL hypersecreting tumor; most common functional pituitary tumor
- excess in male presents as oligospermia or impotence or both
- excess in female causes alterations in infertility

C. GONADOTROPINS: FSH and LH

- for evaluation of fertility and menstrual cycle disorders
- present in serum of both men and women

>The Reproductive System

• Testes: male reproductive organ mainly for production of sperm and steroid hormones
-Testosterone is the predominant hormone produced by the testes; principal
androgen hormone in blood
- testosterone enters the cell and converts dihydrotestosterone (DHT) which
binds to intracellular receptor protein; it influences protein synthesis, cell
growth and development of male secondary sex hormones
- FSH induces Sertoli cells to synthesize androgen binding protein for keeping up
elevated testosterone levels necessary for spermatogenesis
-LH induces Leydig cells to synthesize testosterone

• Ovaries : paired organs for gamete(ovum) and steroid hormone production

-Estrogens promote breast, uterine and vaginal development
>Forms: E1 – Estrone: keeps a healthy thin uterine in menopaused women
E2 – Estradiol: principal estrogen; major form in menstruating women
E3 – Estriol: for healthy thick uterine lining during pregnancy
-Progesterone is produced by corpus luteum; prepares the endometrium for the
implantation of embryo
-Androgens androstenedione, dehydroandrostenedione, testosterone, and
Dihydrotestosterone are produced by the ovaries; excess production leads to
hirsutism(excess hair growth) and loss of female secondary sex characteristics

>Disorders of Sexual Development

1. Hypergonadotropic hypogonadism
Male: Low testosterone, elevated FSH or LH, and impaired sperm production
52
 -Klinefelter’s syndrome: small and firm testicles, gynecomastia(enlargement of male
breast), and azospermia resulting to sterility
Female: elevated FSH with or without LH elevation; results to ovarian failure
-Menopause: ovarian failure that naturally occurs between tha age of 45 to 55
-Primary hypogonadism: premature ovarian failure before the age of 45

2. Hypogonadotropic hypogonadism
Female: deficiency of FSH and LH leading to decreased levels of sex hormones
-common cause of secondary ammenorhea( absence of menses)
Male: low testosterone with low or inappropriately normal FSH or LH
-Kallman’s syndrome: hypogonadism during puberty; presents with anosmia and
midline defects (cleft palate and lip)

3. Polycystic Ovarian Syndrome (PCOS)

-presents as infertility, hirsutism, chronic anovulation, glucose intolerance,hyperlipidemia or
dyslipidemia, and hypertension

D. THYROID STIMULATING HORMONE (TSH): Thyrotropin

- main stimulus for uptake of iodide by the thyroid gland
- made of α and β subunits; α subunit is identical to LH, FSH and HCG αsubunits; β
subunit confers identity of the hormone

>Thyroid Gland/ the butterfly organ: produces thyroid hormones and calcitonin
- Calcitonin is secreted by the parafollicular cells and is involved in calcium regulation
-Parathyroid glandsare located posterior to thyroid gland and involved in calcium regulation
- thyroid follicles colloid thyroglobulin tyrosine
- Iodine is the most important element in thyroid hormone biosynthesis
- Triiodothyronine (T3) : biologically active; mainly produced from tissue deiodination of T4
Reference Value: 60 – 16- ug/dL (0.9 – 2.46 nmol/L)
-Tetraiodothyronine (T4): major fraction of thyroid hormone circulating in blood
Reference Value: 5.5 – 12.5 ug/dL (71 – 161 nmol/L)
-Effects of thyroid hormone include tissue growth, brain maturation, increased heat production,
increased oxygen consumption, and an increased number of beta-adrenergic receptors

>Disorders of the Thyroid

Hypothyroidism Hyperthyroidism
- Low FT4with normal or high TSH - Excess of circulating thyroid hormone
Signs and Bradycardia, coarsened skin, thining Tachycardia, tremor, warm, moist, and smooth
Symptoms of brows, periorbital edema, slowed skin, opthalmopathy, goiter, muscle wasting and
movements, cold intolerance, weakness, nervousness, irritability, anxiety,
menorrhagia, constipation, weight palpitations, weight loss, heat intolerance,
gain, pubertal delay and growth prominence of eyes and fatigue
retardation in children. a. Thyrotoxicosis: result of excessive thyroid
Types a. Primary: Thyroid dysfunction hormone ingestion, leakage of stored hormone
or excessicve production

53
 * Hashimoto’s disease – most b. Grave’s disease: most common cause of
common cause of primary Thyrotoxicosis
hypothyroidism - autoimmune disease with antibodies that
- autoimmune disease that targets the activate the TSH receptor
thyroid gland
- positive for TPO antibody testing

b.Secondary: Pituitary dysfunction

c. Tertiary: Hypothalamic dysfunction
Lab T3/ T4 TSH TRH T3/T4 TSH TRH
Results
Primary decreased increased Increased Primary increased decreased decreased
Grave’s
Secondary decreased decreased Increased Secondary increased increased Decreased
Tertiary decreased decreased decreased Tertiary increased increased Increased

>Thyroid Function Tests:

1. Test for TSH: most useful for assesing thyroid function
• Second generation TSH assay – immunometric
• Third genertion TSH assay – chemiluminiscence

2. serum measurement of T3 and T4 : radioimmunoassay (RIA), chemiluminometric assay, or similar

immunometric technique

3. Thyroglobulin measurments: doubleantibody RIA, enzyme-linked immunoassay (ELISA),

immunoradiometric assay (IRMA), and immunochemiluminescent assay (ICMA) methods

4. T3 uptake: measure amount of available binding sites of TBG; reflects level of TBG
- inverse realtionship: decrease uptake = increase TBG binding sites
- direct relationship with T3 and T4

5. Fine-needle aspiration (FNAB) : most accurate tool for evaluation of thyroid nodules

E. ADRENOCORTICOTROPHIC HORMONE (ACTH): regulates adrenal androgen synthesis

>Adrenal gland: produces steroid hormones and neuropeptides essential for life
Parts of adrenal cortex:
a. Zona Glomerulosa (G zone) : outermost; synthesize mineralocorticoids
b. Zona Fasciculata (F zone) : synthesis of glucocorticoids such as cortisol
c. Zona Reticularis (R zone): innermost; sythesis of androgen DHEAS
>Hormones
1. Aldosterone: chief mineralocorticoid; promotes reabsorption of sodium and water by
kidneys to maintain blood pressure and tonicity
-its secretion is regulated by the RAAS

>Clinical disorders

54
 Hyperaldosteronism Hypoaldosteronism
Types
Primary - Conn’s disease - Result of destruction of
- Aldosterone secreting tumor of adrenal gland, chronic heparin
the adrenals therapy and G-layer enzyme
Screening test: plasma aldosterone/ defficiency
plasma renin activity ratio - Presents with hyperkalemia
Confirrmatory: Saline suppression and metabolic acidosis
test
Secondary - Excessive production of renin
- Results to hypokalemia

2. Cortisol: major Glucocorticoid; maintain blood glucose by inducing lipolysis and amino acid release
from muscle breakdown for conversion to glucose

>Clinical Disorders
Hypercortisolism Hypocortisolism
Over production of CRH or - Adrenal insufficiency
ACTH, adrenal glucocorticoid secondary to adrenal
secretion and exogenous intake destruction or ACTH
- Cushing’s Syndrome: group deficiency
of metabolic d/o - Addison’s disease: primary
characterized by hycortisolism
adrenocortical
hyperfunction
Signs and Symptoms Hypertension, hyperglycemia, Hypotension, hypoglycemia,
central obesity and weakness weight loss and weakness
Testing Screening: overnight Screening: ACTH Stimulation
dexamethasone suppression Test
test Confirmatory: Insulin Tolerance
Confirmatory: low dose Test
dexamethasone suppression
test

3. Weak Androgens: DHEA – principal adrenal androgen; are precursors for production of more
potent androgens and estrogens

>Adrenal Medulla: made of chromaffin cell that secrete catecholamines

-Vanillylmandelic acid (VMA) is the major metabolite of catecholamines except for
dopamine (homovanillylmandelic acid [HVA])
>Hormones
1. Norepinephrine: principal amine
-a neurotransmitter in CNS and in sympathetic nervous system
2. Epinephrine: flight or fight hormone
-most abundant medullary hormone
-Norepinephrine serves as its precursor

55
 - released in response to physical and psychological threats
>Clinical disorders
Pheochromocytoma
Catecholamine producing tumor
Signs and symptoms: Sustained or paroxysmal hypertension
Screening: measurement of plasma total catecholamines and urine metanephrines
Diagnostic: measurement of fractionated metanephrines and catecholamines in a 24-hour
urine

>Hormones Secreted by the Posterior Pituitary gland

A. Antidiuretic hormone (ADH)/ Arginine Vasopressin
- maintain osmotic homeostasis by regulating water balance
- Stimulates V2 receptors on the collecting ducts of the kidneys. It induces increase
production of cyclic AMP (cAMP), which increases permeability and reabsorption of
the water channels (aquaporins).
- modulated by changes in serum osmolality and alterations in intravascular
volume
- maximally stimulated when serum osmolality is >295 mOsm/kg, and supressed
when it falls below 284 mOsm/Kg

>Clinical Significance
A. Diabetes Insipidus (DI): passage of large volumes of dilute urine(>2.5 L/day) but the plasma
osmolality is inappropriately high
• Central: absent of decreased secretion of ADH from the hypothalamus
• Nephrogenic: Renal resistance to ADH action
Diagnostic Test: Water Deprivation Test

B. Syndrome of inappropriate secretion of ADH (SIADH): euvolomic hypoosmolar hyponatremia

Causes: CNS disease, Neoplasm and Pulmonary infections

B. Oxytocin: Stimulated by the stretching of the cervix and vagina during partuition
- contributes to uterine contractions late in labor
- stimulates mammary gland to contract resulting milk ejection (suckling)
- inhibited by stressful situations

Miscellaneous Hormones
1. HUMAN CHORIONIC GONADOTROPIN (HCG)
- synthesized and secreted by the trophoblast cells of developing placenta
- dimeric protein hormone; α subunit is similar to LH, FSH, and TSH
- levels increase at first trimester, but declines at the end of first trimester
- consistently increased HCG indicates: down syndrome, choriocarcinoma and
molar pregnancy

2. Gastrin: secreted by G cells of the stomach

56
 - stimulates parietal cells to secrete HCl
3. Serotonin: amine hormone derived from tryptophan; synthesized by argentaffin cells
- marker for carcinoid tumor

4. Inhibin A: inhibits FSH activity

SPECIALTY AREAS OF CLINICAL CHEMISTRY

PART IV: THERAPEUTIC DRUG MONITORING (TDM): involves the analysis, assessment, and evaluation of
circulating concentrations of drugs in serum, plasma, or whole blood
-its main goal is to ensure that a given drug dosage produces maximal therapeutic benefit and minimal
toxic adverse effects
- common feature of all aspects of TDM is the quantitative evaluation of circulating concentrations of
drugs

>Routes of Administration: intravenous(IV), intramuscula(IM), oral, subcutaneous, inhalation, suppository

and transcutaneous
• Circulatory System: convenient route for effective delivery of most drugs to their site of action
-it offers 100% bioavailability( tha unchanged fraction of the administered dose as it enters
systemic circulation)
• Oral administration is the most common route of administartion; Rectal (suppository) most
commonly used in infants

>Drug disposition: Factors that influence concentration of drugs in the serum

a. Liberation: release of drug
b. Absorption: from site of administration to site of action
c. Distribution: from blood to different parts of the organs
d. Metabolism: modification of drugs
e. Excretion

>Definition of Terms
a. Pharmacokinetics: mathematic modelling of drug concentration in blood circulation; what the body does
to the drug

b. Pharmacodynamics: relationship between the drug conc at the target site and response of the tissue;
what the drug does to the body

c.First-pass effect: : Extensive metabolism of a drug with a high hepatic extraction rate by the liver before it
reaches the systemic circulation

d. Pharmacogenetics: study of the influence of genetic variation on drug response in patients by correlating
gene expression or single-nucleotide polymorphisms with a drug's efficacy or toxicity

e. Pharmacogenomics: relationships and correlations between genetic variation and response or toxicity
associated with drug therapy (pharmacology and toxicology)

f. Therapeutic index: ratio between the minimum toxic and maximum therapeutic serum conc

57
 >Sample collection
-Timing of collection: single most important factor in TDM
- Trough concentration are collected right before the next dose; Peak concentration are colleted
1 hour after administration of drug
-serum or plasma is the specimen of choice; do not use tubes with serum separator gel because
some drugs tend to be absorbed in the gel

A. Cardioactive drugs
Cardiac glycosides
1. Digoxin: for treatment of atrial arrythmias and congestive heart failure
- it blocks rapid atrial conduction signals to the ventricles, thereby slowing
ventricular response
-it also inhibits membrane Na-K-ATPase pumps
-range of therapeutic level: 0.5 – 2 ng/ml
-toxic level: >2 ng/ml
Others
2. Procainamide: treatment of supraventricular or ventricular arrythmias
-therapeutic range: 4 -10 mcg/ml
-toxic level: >12 mcg/ml

3. Quinidine: a naturally occuring drug; purpose similar with procainamide

-therapeutic range: 2.3 – 5 ug/ml
-toxic level: >5 ug/ml

4. Lidocaine: also an antiarrythmic drug; can also be used as anesthesia

- for acute control and prevention of cardian arrythmias

5. Propranolol: beta receptor blocking drug; reduces heart rate, myocardial contractility and output, and
cardian automaticity
-also used as a treatment for angina pectoris, hypertension, and symptomatic coronary artery disease

5. Disopyramide: also for treatment of cardiac arrythmia; used as quinidine substitute

6. Verapamil: blocks activated and inactivated calcium channels

-indicated in hypertension, angina, and supraventricular arrythmias

7. Amiodarone: structural analog of thyroid hormone; blocks potassium channels in cardiac muscle

B. Anticonvulsants
-used for treatment of seizure d/o (grand mal, petit mal, and psychomotor seizures)
- generally(except phenobarbital) they block sodium influx into neurons that have damaged
Membranes

1. Phenobarbital: long-acting barbiturate, used in the treatment of generalized grand mal tonic-
clonic seizures and simple partial seizures, and for anxiety and insomia

58
 -therapeutic range: 15 -30 ug/ml
-Primidone is its inactive form

2. Phenytoin (Dilatin): for treatment of generalized tonic-clonic, simple partial, and complex
partial seizures
-also used as short-term prophylactic agent in brain injury to prevent loss of function
-therapeutic range: 10 – 20 umcg/ml
-toxic level: >20 umcg/ml

3. Primidone: purpose similar to phenytoin; chemical structure is closely related to barbiturates

4. Ethosuximide: drug of choice for absence seizures unaccompanied by other types of seizures
- prefered over valproic acid, at least initially

5. Carbamazepine(Tegretol): primary antiepileptic drug used in the treatment of generalized

tonic-clonic seizures and simple partial and complex partial seizures
-it has serious toxic effects so it is infrequently used

6. Valproic Acid (Depakene): for treatment of generalized tonic-clonic seizures, absence

- seizures, myoclonic seizures, and atonic seizures
-has been shown to have teratogenic effects in experimental animals
C. Antiasthmatics
-Asthma is a form of Chronic Obstructive pulmonary disease that have a variety of causes, and
some of them allergenic in nature

1. Theophylline: used as bronchiodilator for treatment of moderate or severe asthma, both for
prevention of attacks and for treatment of symptomatic exacerbations
-therapeutic range: 10-20 mcg/ml

D. Antiinflammatory and Analgesic Drugs

1. Aspirin: Acetylsalicylic acid
-a nonsteroidal antiinflammatory compound that is used as an analgesic, antipyretic,
and antiinflammatory but in larger doses
-exhibits anticoagulant property due to its antiplatelet activity through inhibiton of
cyclooxygenase (COX) pathway
in platelets, resulting in blockade of platelet plug formation

2. Acetaminophen (Tylenol): used as an analgesic and antipyretic to treat fever, headache, and
mild to moderate myalgia and arthralgia
-prefered over aspirin in patients with bleeding disorder or in children requiring only
antipyretics or analgesics
-hepatotoxic drug in very large doses

E. Immunosuppressives
1. Cyclosporine: maximum suppression occurs during the first 24 hours of antigen stimulation by
the allograft

59
 -indicated to prevent organ rejection in kidney, heart and liver allogenic transplants
-drug of choice for maintenance of the said allografts

2. Tacrolimus(FK-506): a macrolide lactone antibiotic with a mechanism of action similar to

Cyclosporine
-more potent than cyclosporine in its inhibitory effect
- used to prevent transplant rejection

3. Rapamycin (Sirolimus): an antibiotic similar to tacrolimus

4. Mycophenolate Mofetil: a derivative of mycophenolate acid which is a fungal antibiotic

-used as prophylaxis of renal allograft rejection, usually in combination with a steroid or
a calcineurin inhibitor

F. Drugs used in treatment of Mania and Depression

1. Lithium: available comercially as citrate and carbonate salts
-antimanic agent and is used for prophylaxis and treatment of bipolar d/o (manic-
depressive psychosis) and as an aide to antidepressant therapy in melancholic
depression

2. Tricyclic antidepressants: class of drugs used to treat depression, insomnia, extreme apathy,
and loss of libido
-imipramine, amitriptyline, and doxepin are the most relevant

G. Chemotherapeutic agents
1. Methotrexate: inhibits DNA synthesis in all cells
-The efficacy of methotrexate therapy is dependent on a controlled period of inhibition,
one that is selectively detrimental to neoplastic cells

2. Busulfan: an alkylating agent used to treat a variety of leukemia and lymphomas before bone
marrow transplantation
-cytotoxic to marrrow cells and used in combination with cyclophosphamide
-low therapeutic index and potentially cause fatal complications

Part V: THE DRUGS OF ABUSE

1. Cocaine: derived from coca plant; derivative of ecgonine

-normal route of administration: nasal (inhalation or snorting)
- crack is a potent form of cocaine
- medically used to induce local anesthesia during nasopharyngeal surgery
- in high doses it cause euphoric state, induce hallucinatory states and promote violent behavior

2. Opiates ( Heroin, Morphine, Codeine and Fentanyl)

• Morphine: metabolite of heroin; used as powerful analgesic
• Codeine: used as mild analgesic and as an antitussive
• Heroin: induces a pleasant euphoric state; diacetyl form of morphine

60
 • Fentanyl: 80 times more potent than morphine in blocking pain

3. Methadone: administration of this compound allows one to experince the effects of heroin but only in
modulated manner

4. Amphetamines: bear a resemblance to norepinephrine and epinephrine

-cause euphoria and increased mental alertness

5. Tryptamines: derivatives of serotonin

-DMT is one type of tryptamine: also termed as Businessman’s lunch
-Psilocybe: magic mushroom

6. Piperazines: were used as atihelminthics during 1950s

7. Benzodiazepines: VALIUM is the most prominent among this class

-used therapeutically as minor tranquilizers; use to produce calming effects

8. Cannabis: MARIJUANA- one of the oldest and most widely used mind-altering drugs
-a mixture of cut, dried and ground portions of hemp plant Cannabis sativa
-Hashish is a form of more potent product from extraction of the resin of the plant
-δ-9-tetrahydrocannabinol is the principal psychoactive metabolite of marijuana

9. Lysergic acid diethylamide (Lysergide): LSD is a semisynthetic hallucinogen

- one of the most potent pharmacologic materials known producing effects at conc as low as
20mcg and equally effective in injection or oral administration
-it provide the users insights and new ways of solving problems

>Techniques for detecting drugs in serum and urine

Urine is the specimen of choice

Immunoassay methods:
Enzyme-Mediated Immunologic Technique (EMIT)
Fluorescence Polarization Immunoassay (FPIA)
Chromatographic Methods
Thin layer chromatography (TLC): for toxicology screening
High performance Liquid chromatography (HPLC)
Gas Chromatography- Mass Spectrometry (GC-MS): gold standard

Part VI: TOXICOLOGY – The study of poisons

>Four major disiplines: Mechanistic, descriptive, forensic and clinical toxicology
- 50% of poisoning cases is due to suicide attempts; 30% is due to accidental exposure; 20% is
due to homicidal and occupational exposure
-ingestion, transdermal and inhalation are the most common route of exposure

61
 -TD50is the dose that would be predicted to produce toxic response in 50% of the population
-Acute toxicity associated with single, short term exposure to a toxic agent; Chronic toxicity is
asscociated with repeated exposure for extended periods

1. Alcohol: - effects of ROH: disorientation, confusion, and euphoria, which can progress to unconsciousness,
paralysis, and, with high-level exposure, even death

a. Ethanol is the most common drug of abuse

- Ethanol acts as sedative-hypnotic and depresses the CNS irregularly in descending order from cortex to
medulla

b. Methanol: wood alcohol; common solvent

- poisoning occurs in patients who ingest methylated spirits or methanol-containing
Antifreeze

c. Ethylene Glycol: used in car radiator antifreeze

- glycoaldehyde, glycolic acid and glycoxylic acid are the major toxic metabollites

>Blood Alcohol Concentration

Blood ROH conc in % Manifestations
0.01 – 0.05 No obvious impairement; changes in
performance testing
0.03 – 0.12 Mild euphoria; impairement of motor skills
0.09 – 0.25 Loss of critical judgement, memory
impairement and decreased reaction time
0.18 – 0.30 Mental confusion, dizziness, staggering and
slurred speech
0.27 – 0.40 Unable to stand or walk and impaired
conciousness
0.35 – 0.50 Possible comma and death
*≥0.10 = Evidence of Driving under influence of alcohol

>Common indicators of Ethanol abuse

Test Comments
GGT Increases can be seen before the onset of the
pathologic consequences.
Increases in serum activity can occur in many
non-ethanol-related conditions
AST Increases in serum activity can occur in many
non-ethanol-related conditions
AST/ALT ratio A ratio of greater than 2.0 is highly specific for
ethanol-related liver disease
HDL High serum HDL is specific for ethanol
consumption
MCV Increased erythrocyte MCV commonly seen with
excessive ethanol consumption.
62
 Increases are not related to folate or vitamin B12
deficiency

➢ Other Toxic Agents

Carbon Monoxide - From incomplete combustion of carbon-containing subtances
- Intoxication produces tissue hypoxia as a result of decreased
oxygen transport
- Intoxication usually manifests through respiratory, neurologic
and cardiac symptoms with dyspnea as the major symptom
- A cooximeter is used to measure blood carbon monoxide
concentration
Cyanide - A supertoxic compound that can exist as gas, solid or in
solution
Odor of Bitter almonds - Binds avidly to iron in the ferric or trivalent state
- Produces tissue and cellular hypoxia
- Component of some insecticides and rodenticides
- Also produced from burning plastics
- Binding to mitochondrial cytochrome oxidase causes an
uncoupling of oxidative phosphorylation
- ISE and photometric analysis are the most common methods
for evaluation

Arsenic - Used in ant poisons, rodenticides, herbicides and weed killer,

in paints, ceramics and wood presevatives
Odor of garlic - Readily absorbed through the GI tract and lungs
- Arsine gas is the most dangerous form of arsenic
- it binds to proteins resulting to a change in structure and
function
- AAS is the most common method of analysis
- Blood and urine: sample for short term exposure
- Hair and nails: sample for long term exposure
Mercury - Potent enzyme inhibitor; results to structural and functional
change in protein
- Elemental mercury exist in liquid form at room temperature
- Inhalation and ingestion(from contaminated food) are the
primary route of exposure
Lead - Common environmental contaminant
- Ingestion of contaminated dairy products is the most common
route of exposure
- It blocks the activity of delta aminolevulinic acid (ALA)
synthetase
- Toxicity results to appearance of basophilic stippling in RBCs
- Method of analysis include anodic stripping voltametry
Cadmium - Mainly used in electroplating and galvanizing
- Commonly encountered during mining
- Excessive exposure commonly occurs through inhalation and
by ingestion of contaminated food
- Toxic effects are due to its ability to bind proteins
- Causes renal tubular dysfunction
- Evaluation is usually done by AAS
63
 APPENDIX
BASE SI UNITS

Base Quantity Name Symbol

Length Meter m
Mass Kilogram kg
Time Second s
Electric current Ampere A
Thermodynamic temperature Kelvin K
Amount of substance Mole mol
Luminous intensity Candela cd
0
Celsius temperature Degree celsius C
Catalytic activity Katal kat

CONVERSION FACTORS
ANALYTES CONVERSION FACTOR CONVENTIONAL UNIT TO SI UNIT
Glucose 0.0555
Cholesterol
Triglyceride
0.026
0.0113 mg/dl to
BUN 0.357
Calcium
Uric Acid
Phosphorus
0.25
0.0595
0.323
mmol/L
Bilirubin 17.1
Creatinine 88.4 mg/dl to umol/L
Iron 0.179
Albumin
Total Protein 10 g/dl to g/L
Phospholipid 0.01
Bicarbonate
64
 Chloride
Potassium
Sodium 1 mEq/L to
Magnesium 0.5
mmol/L
Lithium 1 mEq/L to umol/L
Ammonia 0.587 ug/dl to umol/L
FT4 ng/dl to pmol/L
Total T4
12.87 ug/dl to nmol/L
FT3 pg/dl to pmol/L
Total T3 0.0154 ng/dl to nmol/L
Amylase 1.85 Somogyi unit to IU/L
Lipase 278 Cherry Crandal to IU/L
Osmolality 1 mOsm/kg to mmol/L
Aldosterone 0.0277 ng/dl to nmol/L
pCO2
pO2 0.133 mm/Hg to kPa
LABORATORY SAFETY

Class of fire Type of Hazard Type of Extinguisher

A Ordinary Combustibles: Wood, Pressurized Water
Paper, Cloth, etc. Dry Chemical
B Flammable Liquid Grease Dry Chemical
Gasoline Paints Oils, etc Carbon Dioxide
C Electrical equipment Motors Carbon Dioxide
Switches Halon
Dry Chemical
D Flammable metals Metal X
Magnesium
E Detonation (Arsenal Fire) Allowed to burn out
K Cooking oils and greases such as Extinguisher especially made for
animals’ fats and vegetable fats. class K fire

65
 https://www.google.com.ph/searcg?q=Nfpa+triangle&oq=Nfpa+triangle&aq=mobile-gws-
lite..&tbm=isch&ved=2ahUKEwjA08jsq8DbAhWCFLwKHVjuC0IQ2-cCegQIABAB7ds=i#imgrc=6Ad3icVgXysEM

>Spills – absorb spills with paper towel, gauze pads or tissue; clean site with common aqueous solution
and disinfect site with 10% bleach then rinse with water

>Chemical safety: MATERIAL SAFETY DATA SHEET (MSDS) - major source of safety information for
employees who may use hazardous materials in their occupations; they are obtained from the chemical
manufacturer
Chemical spills: first step should be to assist/evacuate personnel, and then confinement and
cleanup of the spill can begin

ANALYTICAL CHEMICALS
• Analytic reagent: suitable for use for most analytical procedures; percentage of impurities is
placed on the label
• Ultrapure chemicals: have been put through additional purification steps
-for use in specific procedures such as chromatography, atomic absorption,
immunoassays, molecular diagnostics, standardization, or other techniques that require
extremely pure chemicals

• USP AND NF grade chemicals: used in drug manufacturing

• Chemically pure: impurity limitations not stated and preparations not uniform; not
recommended for use in clinical lab procedures

• Technical or commercial grade: used primarily in manufacturing but never in clinical laboratory

REFERENCE MATERIAL
• Primary Standard: a highly purified chemical that can be measured directly to produce a
substance of exact known concentration and purity
• Secondary standard: a subs of lower purity with concentration determined by comparison with
a primary standard

WATER SPECIFICATIONS
• Water is the most frequently used reagent in the laboratory
• Distilled water: solely purified by distillation; purified to remove almost all organic materials
-Distillation is the process by which a liquid is vaporized and condensed and is used to purify or
concentrate a substance or separate a volatile subs from a less volatile subs
• Deionized water: purified by ion exchange; some or all ions were removed but organic materials
may still be present, so it neither pure nor sterile
-Ion exchange is accomplished by passing water through insoluble resin polymers that contain
anion or cation exchange resins
• RO water: produced through reverse osmosis (pumps water across a semipermeable
membrane)
-RO filters remove 95%-99% of organic compounds, bacteria and other particulate matter, and
about 95% of all ionized and dissolved minerals
66
 • Other purification techniques include ultrafiltration, ultraviolet light, sterilization and ozone
treatment
• Reagent grade water: required for laboratory use
• Categorizing water purity:
-Type I water: has the most stringent requirements and suitable for routine lab use; for test
methods requiring minimum interference
-Type II water: acceptable for most analytic requirements, in reagent, standard and quality
control preparation
-Type III water: autoclave wash water; for glassware washing and not for use in analytical
procedures

CLINICAL LABORATORY SUPPLIES

• Thermometers: used in lab setting to monitor the temp in refrigerators, freezers, water baths,
heating blocks, and incubators
• Glassware and Plasticware:
-Borosilicate glass (Pyrex and Kimax) is the most common type of glassware encountered in
volume measurement; has high degree of thermal resistance and low alkali content

- Special alumina silicate glass(Corex) is strengthened chemically rather than thermally; six
times stronger than borosilicate glass; resist clouding and etching

- Low actinic glassware: has high thermal resistance with an amber or red color; for reagents
needing protection from light

- Vycor is acid and alkali resistant; Flint (soda lime) glass is used for disposable materials such as
glass tubing

- Plastic pipete tips are made of polypropylene; it is chemically resistant and can be autoclaved
- polyethylene is widely used in making test tubes, bottles, graduated tubes, stoppers,
disposable transfer pipets, volumetric pipets and test tube racks

- Polycarbonate is used in tubes for centrifugation, graduated cylinders, and flasks

- Polystyreneis a rigid, clear type of plastic that should not be autoclaved; used in making
capped graduated tubes and test tubes

- Teflon: for manufacturing stirring bars, tubing, cryogenic vials, and bottle cap liners; resistant
to acids, bases, alcohols and hydrocarbons

• Volumetric Laboratory ware

a. Pipets
-TC (to contain) are referred to as rinse-out pipets; they contain the exact
amount of liquid that must be completely transferred
Example: Sahli hemoglobin and Long-Levy pipets
-TD (to deliver) are designed to drain by gravity; should not be blown out
Example: Mohr, serologic and volumetric pipets

67
 -Volumetric pipets have an open-ended bulb; allowed to drain freely and not be
shaken, and are used to dispense aqueous solutions
-Ostwald Folin pipets are used for viscous samples
-Mohr pipet does not have graduations to the tip
-Serologic pipet have graduations to the tip

I. DESIGN
A. TO CONTAIN (TC)
B. TO DELIVER (TD)
II. DRAINAGE CHARACTERISTICS
A. BLOWOUT
B. SELF-DRAINING
III. TYPE
A. MEASURING OR GRADUATED
1. SEROLOGIC
2. MOHR
3. BACTERIOLOGIC
4. BALL, KOLMER, OR KAHN
5. MICROPIPET
B. TRANSFER
1. VOLUMETRIC
2. OSTWALD-FOLIN
3. PASTEUR PIPETS
4. AUTOMATIC MACROPIPETS OR MICROPIPETS

b. Volumetric flasks: used in preparing standards and materials requiring optimal

accuracy

c. Automatic pipets: most widely used are the air-displacement (piston operated and
with disposable pipete tips) and positive displacement(use capillary tips and Teflon-
tipped plunger) types; they deliver 1-1000 uL

Basic Separation Techniques

1. Centrifugation –centrifugal force is used to separate solid matter from liquid suspension
- Centrifugal force depend on mass, speed and radius; speed is related to RCF
RCF = 1.118 x 10-5 x r x (rmp)2
-cleaning must be done daily; speed is checked once every 3 months using a tachometer and the
timer is checked weekly against a reference timer

Types: a. Horizontal-head or Swinging bucket – the buckets start off in a vertical position but during
acceleration of the rotor swing out to a horizontal position. At deceleration, the tube returns to
its original position.

b. Fixed-angle rotors – the tubes are located in holes in the rotor body set at a fixed angle
between 140 and 400 to the vertical.
68
 c. Vertical tube rotors- are zero angle fixed rotors in which tubes are aligned vertically in the
rotor at all times

2. Filtration

3. Dialysis- for separating macromolecules from a solvent or smaller substances

Laboratory Statistics and Quality Management

➢ Quality Control (QC) – process to periodically examine a measurement procedure to verify that it is
performing according to preestablished specifications
• QC samples – are measured periodically in the same manner as clinical samples and their results
examined to ensure that the measurement procedure meets performance requirements
-Two different concentrations are necessary for adequate statistical QC

• QC charts: Levey-Jennings chart - graphically represent the observed values of a control

material over time in the context of the upper and lower control limits; easily identifies errors
a.Trend – control values that either increase or decrease for six consecutive days; due to
reagent deterioration
b.Shift – when control values distribute themselves on one side or either side of the mean for
six consecutive days; due to improper calibration of instruments
c.Outliers – highly deviating values

• Westgard Rules: Multirule procedure to further judge whether control results indicate out-of-
control situations

12S One control observation exceeding the mean ±2S. A warning rule that
initiates testing control data by other rules.
13S One control observation exceeding the mean ±3S. Allows high
sensitivity to random error.
22S Two control observations consecutively exceeding the same +2S or -2S.
Allows high sensitivity to systematic error.
R4S One control exceeding the +2S and another exceeding -2S. Allows
detection of random error • QC
41S Four consecutive control observations exceeding +1S or -1S. This allows results –
the detection of systematic error
10X Ten consecutive control observation falling on one side or the other of
the mean. Allows detection of systematic error.
acceptable reference limit is at ±2SD

➢ Quality management – encompasses all concepts of QC; the entire testing process is directed with the
overall goal improving the accuracy of laboratory results
➢ Quality Assurance - Measurement of the broader dimensions of quality, from the perspective of the end-
users. Involve the monitoring of specimen acquisition, turnaround times, or proficiency testing of materials
to determine analytic performance
➢ Quality Improvement – its purpose is to determine and address the root causes of problems identified by
QC and quality assurance
69
➢ Delta Check –comparing a patient’s current test result against a previous result for the same analyte
➢ Proficiency testing (external quality assessment) – evaluation of method performance by comparison of
results versus those of other labs for the same set of samples
➢ Descriptive statistics – provides a consistent framework for calculating or estimating the central tendency
of continuous data in forms of mean, median, and mode.
-they describe what the magnitude of results is and how the data points differ from one another
• Central Tendency: MEAN – average value; Mean = (x1 + x2 + x3+…xn) ÷n
• Median - the middle of the data after the data have been rank ordered
• Mode - the most frequently occurring value in a dataset
➢ Gaussian (normal) distribution – a symmetrical, bell-shaped curve centered about the mean value
• The area under the curve is 1.0 or 100%
• “68-95-99 rule” summarizes the relationships between the area under a gaussian distribution and
the standard deviation

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➢ Standard Deviation (SD) – common parametric measure of the dispersion of data points about the mean
value of a population under examination
➢ Coefficient of Variation – SD divided by the mean; expressed as percentage
➢ Variance – SD squared; measure of variability
➢ t-Test – most common method of comparison of mean values between groups
➢ F-test – method of comparison of SD between groups
➢ Parameters of QC:
• Accuracy – ability to correctly detect and quantify an analyte; nearness or closeness of assayed
value to true value
• Precision – reproducibility of an assay; ability to produce repeated results that agree with one
another
• Analytic sensitivity – lowest value that an assay can reliably detect
• Analytic specificity – ability of method to measure only the analyte of interest

VARIATIONS
➢ Random error –present in all measurements and can either be positive or negative; due to chance
Result of: instrument, operator, reagent, and environmental variations; pipetting errors, mislabeling, temp
fluctuations and improper mixing
➢ Systematic error – influences observation consistently
Result of: calibration problems, reagent deterioration, improperly made standards solutions, contaminated
solutions and unstable reagent blanks
➢ Clerical errors –occurs in handwritten labels and result forms

Stages of Analytic Testing

➢ Preanalytical phase – focuses on sample processing
70
➢ Analytical phase – focuses on chemical analyses
➢ Postanalytical phase – focuses on data management (Result forms)

Preanalytical errors Analytical errors Postanalytical error

Physiologic factors Instrument malfunction Inappropriate validation
- Diurnal variation Incorrect sample loaded of data
- Exercise and diet Interfering substances in Failure to report or report
- Posture, age and gender the assay sent to wrong location
Specimen collection Undetected QC failure Lengthy turnaround time
- Patient misidentification Data entry/ transcription
- Specimen mislabeling error
- Short draws/ wrong anticoagulant used Critical value not reported
- Mixing error/ clots Results incorrectly
- Hemolysis/ lipemia interpreted
- Hemoconcentration
- Exposure to light/ extreme temp
- Improperly timed specimen
- Missed or incorrectly interpreted lab
requests
- Inappropriate test ordered
- Pipetting and centrifugation error

ANALYTICAL TECHNIQUES
1. Spectrophotometry – used to measure light transmitted by a solution to determine the concentration of the
light-absorbing substance in solution
➢ Definition of Terms:
• Electromagnetic radiation – photons of energy traveling in waves
• Frequency – number of oscillations of the waveform per second
• Wavelength – distance between two consecutive peaks; expressed in nanometers
400-700 nm = visible spectrum <400 nm = UV >700nm = Infrared

* Frequency is inverse to wavelength

Wavelength (nm) Region Name Observed
<380 Ultraviolet Invisible
380-440 Violet
440-500 Blue
500-580
580-600
Visible Green
Yellow
600-620 Orange
620-750 Red
800-2500 Near-infrared Not visible
2,500-15,000 Mid-infrared Not visible
15,000- 1,000,000 Far-infrared Not visible

• Beer-Lambert Law – A = 2 – log %T

- relationship between absorption of light by a solution and the concentration of that solution
-absorbance is directly proportional to concentration and inversely proportional to
transmittance
71
 ➢ Components of spectrophotometer
A. Light Source-provides energy that the sample will modify by absorption; light is polychromatic
• Tungsten or Tungsten-Halogen lamps – widely used for testing in the visible region
• Deuterium lamp – provides UV radiation in analytic spectrophotometers; provides continuous
emission down to 165 nm
• Mercury-arc lamp – also used UV work.

B. Monochromators – for isolation of individual wavelengths of light

• Interference Filters - produce monochromatic light based on the principle of constructive
interference of waves
• Prism
• Diffraction grating – most commonly used; has parallel grooves etched onto a polished surface
- based on the principle that wavelengths bend as they pass a sharp corner
C. Sample cell –used to hold samples and must be made of materials that is transparent to
radiation in spectral region of interest
• Quartz cuvets – used for applications requiring UV regions
• Alumina Silica – most commonly used

D. Photodetectors - convert the transmitted radiant energy into an equivalent amount of electrical
energy
• Barrier-layer cell/ photocell – used for detecting and measuring radiation in the visible region
• Photomultiplier tubes – most common and is used when radiation power is low; detects and
amplifies radiant energy
• Photo tube – similar to photocell except that it requires an outside voltage for operation

➢ Types
a. Single-beam – simplest type; designed to make one measurement at a time at one specified
wavelength

b. Double-beam – splits or chops the monochromatic beam of radiation into two components. One
beam passes through the sample and the other through the reference solution
• Double- beam in space – uses two photodetectors
• Double-beam in time – uses one photodetector and alternately passes the monochromatic
radiation through the sample cuvet and to the reference solution using a chopper
•
c. Atomic Absorption Spectrophotometer - used to measure concentration by detecting absorption of
electromagnetic radiation by atoms rather than by molecules
- routinely used to measure concentration of trace metals that are not easily excited

2. Flame Photometry - measures light emitted by excited atoms

3. Fluorometry– works under the principle that luminescence is based on an energy process that occurs when
certain compounds absorb electromagnetic radiation, become excited and return to an energy level lower than
or equal to their original level
- it is a specific and sensitive technique; high sensitivity to environmental changes is its greatest
Disadvantage
72
4. Chemiluminiscence - part of the chemical energy generated produces excited intermediates that decay to a
ground state with the emission of photons
- The emitted radiation is measured with a PM tube, and the signal is related to analyte concentration

5. Nephelometry– light scattered by particles is measured at an angle, typically 15-90 degrees to the beam
incident on the cuvet
- light scattering depends on the wavelength of the incident light and particle size

6. Turbidimetry – measurement of the reduction of light transmission caused by particle formation

- amount of light absorbed depends on the specimen concentration and particle size
7. Reflectometry – used in urine dipstick analysis and dry slide chemical analysis
8. Electrochemistry
a. Potentiometry – measurement of potential (voltage) between two electrodes in a solution
b. Coulometry – measures the quantity of electricity needed to convert an analyte to a different
oxidation state
c. Amperometry – measurement of the current flow produced by an oxidation-reduction reaction
d. Voltametry – method in which a potential is applied to an electrochemical cell and the resulting
current is measured

9. Electrophoresis- migration of charged solutes or particles in an electrical field

*Iontophoresis:migration of small ions
*zone electrophoresis: migration of charged macromolecules in a porous support medium such
as paper, cellulose acetate, or agarose gel film
*Capillary electrophoresis: separation is performed in narrow-bore fused silica capillaries
-the greater the net charges of a dissolved compound, the faster it moves through the solution toward the
oppositely charged electrode

➢ Common support media: cellulose acetate, agarose, and polyacrylamide gels

-once electrophoresis is completed, the support medium is treated with a dye to identify separated fractions
➢ Common dyes: Amido Black, Ponceau S, Fat Red 7B, and Sudan Black B

10. Densitometry- measures the absorbance of the stain on a support medium (from electrophoresis)

11. Isoelectric Focusing–modification of electrophoresis

-charged proteins migrate through a support medium that has a continuous pH gradient

12. Chromatography – separation method based on different interactions of the specimen compounds with the
mobile phase and with the stationary phase as the compounds travel through a support medium
-classified according to their mobile phase

a. Gas Chromatography (GC) - useful for compounds that are naturally volatile or can be easily
converted to volatile forms.
-has high resolution, low detection limits, accuracy and short analytic time

b. Liquid Chromatography (LC) –use lower temp for separation, thereby achieving better separation of

73
 thermolabile compounds
• High performance Liquid Chromatography (HPLC) - uses pressure for fast separations, controlled
temperature, in-line detectors, and gradient elution techniques

c. Thin layer Chromatography (TLC) - most commonly used as a semiquantitative screening test

12. Mass Spectrometry (MS) – based on fragmentation and ionization of molecules using suitable source of
Energy

-the resulting fragment masses and their relative abundance yield a characteristic mass spectrum of the
parent molecule
-compounds for MS must be isolated first by GC or HPLC
-used for quantitation and identification of compounds; for determining structural information and
molecular determination

13. Osmometry – measurement of the osmolality of an aqueous solution

*osmotically active particles: Glucose, urea nitrogen and sodium
-colligative properties: osmotic pressure, boiling point, freezing point and vapor pressure
-when the osmotic pressure of a solution increases

!!!!! 1. Osmotic pressure increases

2. Boiling point elevates
3. Freezing point is depressed
4. Vapor pressure is depressed

AUTOMATION
➢ Random access analyzers – stat specimens could be analyzed out of sequence from the batch as
needed.

Approaches to automation
• Continuous flow – liquids are pumped through a system of continuous tubing. Samples are introduced
in a sequential manner, following each other through the same network. A series of air bubbles at
regular intervals serve as separating and cleaning media

• Discrete analysis-is the separation of each sample and accompanying reagents in a separate container;
have the capability of running multiple tests one sample at a time or multiple samples one test at a time
-most popular and versatile analyzers

Compiled by: Shane Marie R. Piamonte, RMT

May 2018 edition

REFERENCES:

74
1. Bishop, Michael L., et.al.(2010). Clinical Chemistry – Principles, Procedures and Correlations. 6th ed.
Lippincott Williams and Wilkins.
2. Burtis, Carl A., et.al. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6thed.
3. Mc Pherson, Richard A. and Matthew R. Pincus. (2011). Henry’s Clinical Diagnosis and Laboratory
Tests. 22nded.Saunders
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