Glucose

- Monosaccharide
- Only carbohydrate to be directly used as source of energy
- CNS is highly dependent on glucose
- Normal level: 70 – 100 mg/dL
o <70- hypoglycemic
o <100-impaired glucose metabolism
o <126- hyperglycemic

Organs that are involved in the maintenance of Glucose Concentration:

 Pancreas- both an exocrine and endocrine gland

o exocrine: production of enzymes
 Amylase (breakdown of complex carbohydrates/lipids)
 lipase
 both are pancreatic marker, these enzymes will elevate
o endocrine: production of hormones
 insulin
 glucagon
 somatostatin
 Liver- supplies/formation glucose
o ex. fasting- conversion of glycogen to glucose: glycogenolysis
o prolonged fasting: gluconeogenesis –conversion of non-carbohydrate molecule
to glucose
 Endocrine glands
o Adrenal gland- cortisol, catecholamines
o pituitary gland- ACTH, Grow hormone
o Thyroid gland- thyroxine (regulation of glucose)

Pancreas:

 Both an _Endocrine and Exocrine gland_

 Production of Enzymes (exocrine): Amylase, Lipase
 Production of Hormones (Endocrine): Insulin, Glucagon, Somatostatin
Insulin

 the primary hormone responsible for the entry of glucose into the cell.
 It is synthesized by the _Beta-cells_ of islets of Langerhans in the pancreas.
 When these cells detect an increase in body glucose, they release insulin. (to counter
the release of glucose)
 The release of insulin causes an increased movement of glucose into the cells and
increased glucose metabolism/breakdown of glucose
 Insulin is normally released when glucose levels are high and is not released when
glucose levels are decreased.
  It decreases plasma glucose levels by increasing the transport entry of glucose in
muscle and adipose tissue
 It also regulates glucose by increasing
glycogenesis- conversion of excess glucose to glycogen
lipogenesis- formation of fat
and glycolysis- breakdown of glucose
and inhibiting glycogenolysis- conversion of glycogen to glucose

 Insulin is the only hormone that decreases glucose levels and can be referred to as a
hypoglycemic agent
Glucagon

 Glucagon is the primary hormone responsible for increasing glucose levels/ primary
hyperglycemic agent (bc there are other hormones that can inc glucose)
 It is synthesized by the Alpha-cells of islets of Langerhans in the pancreas and released
during stress and fasting states.
 When these cells detect a decrease in body glucose, they release glucagon.
 Glucagon acts by increasing plasma glucose levels by glycogenolysis in the liver and an
increase in gluconeogenesis (formation of glucose from non-carbohydrate source)
 It can be referred to as a hyperglycemic agent

Epinephrine

 produced by the adrenal medulla (inner portion of adrenal gland)

 Increases plasma glucose by inhibiting insulin secretion, increasing glycogenolysis, and
promoting lipoliysis.
 Epinephrine is released during times of stress.
Cortisol

 Glucocorticoid – steroid produced by the adrenal cortex that is important for the
maintenance of glucose
 Released from the adrenal cortex on stimulation by adrenocorticotropic hormone
(ACTH). ACTH is produced by the pituitary gland, and is necessary to stimulate adrenal
gland to produce cortisol
 Cortisol increases plasma glucose by decreasing entry into the cell and increasing
gluconeogenesis, breakdown of liver glycogen, and lipolysis.
Growth Hormone

 Its release from the pituitary is stimulated by decreased glucose levels( fasting state)
and inhibited by increased glucose
 increases plasma glucose by decreasing the entry of glucose into the cells and
increasing glycolysis (hyperglycemic agent)
Thyroxine

 increases plasma glucose levels by increasing glycogenolysis, gluconeogenesis, and

intestinal absorption of glucose.
 T4 produced by the thyroid gland
ACTH

 Stimulates the adrenal cortex to release cortisol

 increases plasma glucose levels by converting liver glycogen to glucose (glycogenolysis)
and by promoting gluconeogenesis
Somatostatin

 produced by the Delta-cells of the islets of Langerhans of the pancreas

 increases plasma glucose levels by the inhibition of insulin, glucagon, growth hormone,
and other endocrine hormones.

Hyperglycemia

 is an increase in plasma glucose level.

 In healthy patients, during a hyperglycemia state, insulin is secreted by the b-cells of the
pancreatic islets of Langerhans.
 Insulin enhances membrane permeability to cells in the liver, muscle, and adipose
tissue.
 Hyperglycemia, or increased plasma glucose levels, is caused by an imbalance of
hormones.
 Causes:
1. Stress- body will produce catecholamines
2. severe infection
3. dehydration- drop in plasma volume
4. pregnancy- hormonal imbalance
5. pancreatectomy- removal of a part/whole pancreas, pancreas is important in
the release of insulin, so if it is removed, there is low insulin production
6. hemochromatosis- accumulation of iron in our system/ iron overload and may
accumulate in the pancreas
7. insulin deficiency- autoantibodies
8. abnormal insulin receptor- insulin resistance, cause of type 2 DM
 FBS > 126 mg/dl (you need 2 results of FBS to confirm if it is diabetic)
Diabetes mellitus

 a group of metabolic diseases characterized by hyperglycemia resulting from defects in

insulin secretion (type 1), insulin action (type 2), or both.
Type 1 diabetes

 result of cellular-mediated autoimmune destruction of the beta-cells of the pancreas,

causing an absolute deficiency of insulin secretion
 constitutes only 10% to 20% of all cases of diabetes and commonly occurs in childhood
and adolescence.
 Characteristics of type 1 diabetes include abrupt onset, insulin dependence and ketosis
tendency
 Autoantibodies responsible for the destruction of beta-cells: islet cell autoantibodies,
insulin autoantibodies, glutamic acid decarboxylase autoantibodies, and tyrosine
phosphatase IA-2 and IA-2B autoantibodies
 Signs and symptoms: polydipsia (excessive thirst), polyphagia (increased food intake),
polyuria (excessive urine production), rapid weight loss (inc fat utilization),
hyperventilation, mental confusion, and possible loss of consciousness (due to
increased glucose to brain).
 Complications include microvascular problems such as nephropathy (kidney damage),
neuropathy (nerve damage), and retinopathy (eye damage)
Type 2 diabetes mellitus

 characterized by hyperglycemia as a result of an individual’s resistance to insulin

 This resistance results in a relative, not an absolute, insulin deficiency. you have normal
insulin conc but fewer when compared to the glucose concentration
 Type 2 constitutes the majority of the diabetes cases.
 Most patients in this type are obese or have an increased percentage of body fat
distribution in the abdominal region.
 This type of diabetes often goes undiagnosed for many years and is associated with a
strong genetic predisposition, with patients at increased risk with an increase in age,
obesity, and lack of physical exercise.
 Characteristics usually include adult onset of the disease and milder symptoms than in
type 1, with ketoacidosis seldom occurring.
 However, these patients are more likely to go into a hyperosmolar coma (inc number of
solute in blood) and are at an increased risk of developing macrovascular (heart attack,
stroke) and microvascular complications
Other specific types of diabetes

 associated with certain conditions (secondary), including genetic defects of b-cell

function or insulin action, pancreatic disease, diseases of endocrine origin, drug- or
chemical-induced insulin receptor abnormalities, and certain genetic syndromes.
 pancreatic disorders- hemochromatosis (build-up of iron in pancreas)
 pancreatectomy
 drugs or chemical inducer of beta cell dysfunction- ex glucocorticoid, pentamidine
 genetic syndromes- down syndrome, klienfilter syndrome, rhabson menden hal
syndrome will have insulin resistance causing other type of diabetes
 exocrine disorders- cystic fibrosis
 endocrine disorder- pheochromocytoma (tumor in your adrenal medulla, overproduction
of catecholamines), gigantism, acromegaly
  The characteristics and prognosis of this form of diabetes depend on the primary
disorder.

Gestational Diabetes Mellitus

 defined as any degree of glucose intolerance with onset or first recognition during
pregnancy. (but if the woman is diabetic before preganancy, then that is not GDM)
 Causes of GDM include metabolic and hormonal changes.
 Patients with GDM frequently return to normal postpartum. However, there is an
increased risk for the development of diabetes in later years (DM type 2)
 Infants born to mothers with diabetes are at increased risk for respiratory distress
syndrome, hypocalcemia, and hyperbilirubinemia.
 Fetal insulin secretion is stimulated in the neonate of a mother with diabetes. However,
when the infant is born and the umbilical cord is severed, the infant’s oversupply of
glucose is abruptly terminated, causing severe hypoglycaemia (countered by
introduction of sugar)
 Hypoglycemia in infants of a mother with GDM because of overproduction of insulin, so
when it is stopped, grabe ang pag-use sa glucose because of hyperinsulinism.

Pathophysiology of Diabetes Mellitus

 In both type 1 and type 2 diabetes, the individual will be hyperglycemic, which can be
severe.
 Glucosuria can also occur after the renal tubular transporter system for glucose
becomes saturated. Renal Threshold for glucose: 160-180 mg/dL
 The individual with type 1 diabetes has a higher tendency to produce ketones (product of
fat breakdown)
 Patients with type 2 diabetes seldom generate ketones but instead have a greater
tendency to develop hyperosmolar nonketotic states (ratio of glucose to plasma is too
high/saturated)
 In type 1, there is an absence of insulin with an excess of glucagon. This permits
gluconeogenesis and lipolysis to occur.
 In type 2, insulin is not absent and may, in fact, present as hyperinsulinemia at times;
therefore, glucagon is attenuated.
 Acetoacetate, b-hydroxybutyrate, and acetone are produced from the oxidation of fatty
acids. The two former ketone bodies contribute to the acidosis.
 former names: Type 1= ketosis prone DM, Type 2= ketosis resistant DM
 Bicarbonate(cause) and total carbon dioxide (compensation) are usually decreased due
to Kussmaul-Kien respiration (deep respirations).
 Serum osmolality (Glucose, Sodium, Urea) is high as a result of hyperglycemia; sodium
concentrations tend to be lower due in part to losses (polyuria) and in part to a shift of
water from cells because of the hyperglycemia.
 Hyperkalemia is almost always present as a result of the displacement of potassium
from cells in acidosis.
  More typical of the untreated patient with type 2 diabetes is the nonketotic hyperosmolar
(taas og solute than solvent) state. The individual presenting with this syndrome has an
overproduction of glucose; however, there appears to be an imbalance between
production and elimination in urine.
 Often, this state is precipitated by heart disease, stroke, or pancreatitis.
 Glucose concentrations exceed 300 to 500 mg/dL (17 to 28 mmol/L) and severe
dehydration is present. The severe dehydration contributes to the inability to excrete
glucose in the urine. Mortality is high with this condition.
 Ketones are not observed because the severe hyperosmolar state inhibits the ability of
glucagon to stimulate lipolysis.
 The gross elevation in glucose and osmolality, the elevation in BUN, and the absence of
ketones distinguish this condition from diabetic ketoacidosis.

Criteria for testing Pre-diabetes and Diabetes

According to the ADA recommendations, all adults beginning at the age of 45 years should be
tested for diabetes every 3 years using either the haemoglobin A1c (HbA1c), fasting plasma
glucose, or a 2-hr 75 g oral glucose tolerance test (OGTT) unless the individual has otherwise
been diagnosed with diabetes.
Testing should be carried out at an earlier age or more frequently in individuals who display
overweight tendencies and have additional risk factors, as follows:

 Habitually physically inactive

 Family history of diabetes in a first-degree relative
 In a high-risk minority population (e.g., African American, Latino, Native American, Asian
American, and Pacific Islander)
 History of GDM or delivering a baby weighing more than 9 lb (4.1 kg)
 Hypertension (blood pressure ≥ 140/90 mm Hg)
 Low high-density lipoprotein (HDL) cholesterol concentrations (250 mg/dL (2.82 mmol/L)
 History of impaired fasting glucose/impaired glucose tolerance
o 120- pre-diabetic
o 70-99- Normal
o 100-125- pre-diabetic/ impaired fasting glucose
o > 126- diabetic/ hyperglycemic
 Women with polycystic ovarian syndrome (PCOS)
 Other clinical conditions associated with insulin resistance (e.g., severe obesity and
acanthosis nigricans- blackening at the back of the neck and near the armpits of obese
people)
 History of cardiovascular disease
Testing for prediabetes and diabetes should begin at the age of 10.
Testing should be repeated at least at 2 years intervals, with consideration of more frequent
testing depending on initial results and risk status.
As the incidence of adolescent type 2 diabetes has risen dramatically in the past few years,
criteria for the testing for type 2 diabetes in asymptomatic children have been developed. These
criteria include initiation of testing at the age 10 years or at the onset of puberty, if puberty
occurs at a younger age, with follow-up testing every 2 years.
Type 1- Juvenile onset diabetes mellitus
Type 2- adult onset diabetes mellitus

 but now common in children

Testing should be carried out on children who display the following characteristics:

• Overweight
• Family history of type 2 diabetes in first- or second degree relative
 Race/ethnicity (e.g., Native American, African American, Latino, Asian American, and
Pacific Islander)
 Signs of insulin resistance or conditions associated with insulin resistance (e.g.,
acanthosis nigricans, hypertension, dyslipidemia, and PCOS)
• Maternal history of diabetes or GDM

Criteria for diagnosis of Diabetes Mellitus

(1) HbA1c > 6.5% using a National Glycohemoglobin Standardization Program (NGSP)–
certified method, (2) a fasting plasma glucose > 126 mg/dL
(3) an OGTT with a 2-hour postload (75 g glucose load) level > 200 mg/dL
(4) Symptoms of diabetes plus a random plasma glucose level ≥ 200 mg/dL, (not reliable, so
only 1,2,3 are used in hospitals)
- it is not reliable if you use random plasma twice, but you can do random first and then
confirmed by any of the three above

 each of which should be confirmed on a subsequent day by any one of the first three methods
Any of the first three methods are considered appropriate for the diagnosis of diabetes.
Point-of-care assay methods for either plasma glucose or HbA1c are not recommended for
diagnosis.

 point of care is only for monitoring and determination of right dose of insulin, not for
diagnosis

Categories of Fasting Plasma Glucose

• Normal fasting glucose – 70-99 mg/dL
• Impaired fasting glucose – 100-125 mg/dL
• Diabetes - > 126 mg/dL
Categories of oral Glucose tolerance test
-diagnose GDM
• Normal glucose tolerance ………….. two-hour PG < 140 mg/dL
• Impaired glucose tolerance …………two-hour PG 140-199 mg/dL
• Diabetes ………………………………………two-hour PG > 200 mg/dL
Categories of Glycosylated haemoglobin

 4%- 5.6%- Normal

 5.7%- 6.4%- Pre-diabetes
 > 6.5%- Diabetes

Criteria for testing and diagnosing GDM

• The revised criteria recommend that all nondiabetic pregnant women should be
screened for GDM at 24-28 weeks of gestation.
• The approach for screening and diagnosis is the performance of a 2-hour OGTT using a
75 g glucose load.
• Glucose measurements should be taken at fasting (8hrs), 1 hour, and 2 hours.
Screening and diagnosis:
fast 8hrs-collect blood- drink- 1hr after, collect blood- 2hrs after, collect blood
• A fasting plasma glucose value > 92 mg/dL (5.1 mmol/L), a 1-hour value > 180 mg/dL
(10 mmol/L), or a 2-hour glucose value > 153 mg/dL (8.5 mmol/L) is diagnostic of GDM
if any one of the three criteria are met.
• This test should be performed in the morning after an overnight fast of at least 8 hours

Ketones

 Ketone bodies are produced by the liver through metabolism of fatty acids to provide a
ready energy source from stored lipids at times of low carbohydrate availability
 ketone production if low carbohydrate availability (ex. low glucose, low glycogen)
 acetone (2%), acetoacetic acid (20%), and Beta-hydroxybutyric acid (78%)
 A low level of ketone bodies is present in the body at all times
Increased ketones:

 carbohydrate deprivation- low carb available or no stored glycogen

 diabetes mellitus- px has excess glucose but the cells cannot utilize bc of insulin
deficiency/resistance, so mangita og lain na source of energy w/c is lipids
 starvation/ fasting-
 high-fat diets- ex. keto-diet
 prolonged vomiting
 glycogen storage disease- problems with conversion of glycogen to glucose, so maguse
og lipids
 Ketonemia- accumulation of ketones in blood
Ketonuria- accumulation of ketones in urine

Applications of ketone measurement:

 px with type 1 diabetes during acute illness

 stress
 pregnancy
 elevated blood glucose levels above 300 mg/dL (common in type 2 DM)
 the patient has signs of ketoacidosis
Specimen requirements

 Fresh serum or urine

 The sample should be tightly stoppered and analyzed immediatey
 Gerhardt`s test used in ferric chloride reacted with acetoacetic acid to produce a red
color
 Sodium nitroprusside reacts with acetoacetic acid in an alkaline pH to form a purple
color. If the reagent contains glycerin, the acetone is also detected. This method is used
with the urine reagent strip test and Acetest tablets
 A newer enzymatic method adapted to some automated instruments uses the enzyme 3-
hydroxbutyrate dehydrogenase to detect either Beta-hydroxybtyric acid or acetoacetic
acid

Microalbuminuria
 Diabetes mellitus causes progressive changes to the kidneys and ultimately results in
diabetic renal nephropathy/diabetic kidney disease
 An early sign that nephropathy is occurring is an increase in urinary
albumin/microalbuminuria
 Microalbumin measurements are useful to assist in diagnosis at an early stage and
before the development of proteinuria.
 An annual assessment of kidney function by the determination of urinary albumin
excretion is recommended for diabetic patients
 Microalbuminuria is defined as persistent albuminuria in two out of three urine collections
of 30 to 300 mg/day, 20 to 200 ug/min, or an albumin-creatinine ratio of 30-300 ug/mg
creatinine
 Clinical proteinuria or macroalbuminuria is established with an albumin-creatinine ratio
>300 mg/day, >200 ug/min, or >300 ug/mg.
 Although three methods for microalbuminuria screening are available, the use of a
random spot collection for the measurement of the albumin-creatinine ratio is the
preferred method
 Using the spot method, without the simultaneous creatinine measurement, may result in
false-positive and false-negative results because of variation in urine concentration
  A patient is determined to have microalbuminuria when two of three specimens collected
within a 3-to-6-month period are abnormal
 Factors that may elevate the urinary excretion of albumin include exercise within 24
hours, infection, fever, congestive heart failure (related to Angiotensin II), marked
hyperglycemia, and marked hypertension.

C-peptide test

 Formed during the conversion of pro-insulin to insulin

 Reliable indicator of beta cell function (directly proportional)
 Monitor individual response to pancreatic surgery
 Mainly evaluates hypoglycemia and continuous assessment of beta cell function
 increased: Insulinoma (directly proportional to c-peptide bc of the tumor) ,
Type 2 DM
Type 1 DM= decreased c-peptide
Type 2 DM= increased c-peptide

Islet autoantibody and Insulin Testing

 The presence of autoantibodies to the b-islet cells of the pancreas is characteristic of

type 1 diabetes
 islet autoantibody testing is not currently recommended for routine screening for
diabetes diagnosis
 Insulin measurements are not required for the diagnosis of diabetes mellitus, but in
certain hypoglycemic states, it is important to know the concentration of insulin in
relation to the plasma glucose concentration
 Diagnosis of insulinoma- >8 U/ml of insulin after fasting
ANALYTICAL METHODS
ENERGY
-Is transmitted via electromagnetic waves that are
characterized by their frequency and wavelength.
WAVELENGTH
 Is the distance between two successive peaks
and it is expressed in terms of nanometer
 Visible spectrum: 400-700
 -UV < 400 (violet)
 -Infrared >700 (red)
FREQUENCY
 is the number of vibrations of wave motion per
second
 The lower the wave frequency, the longer the
wavelength
 The wavelength is inversely proportional to
energy
*In terms of energy the ultraviolet > infrared
 Wavelength accuracy is the wavelength
indicated on the control dial is the actual
wavelength
 Wavelength accuracy is the wavelength
indicated on the control dial is the actual of
light passed by the monochromator
 Didymium or holmium oxide filter is used to
check wavelength accuracy
 Neutral density filter and dichromate solution
verify absorbance accuracy and linearity
I. COLORIMETRY
a. Spectrophotometric measurement- is measurement
of light intensity in a narrower wavelength.
*need to select a certain wavelength
b. Photometric measurement- is measurement of light
intensity
*not concerned with a specific wavelength; can use
range
A. SPECTROPHOTOMETRY
-Involves the measurement of light transmitted by a
solution to determine the concentration of the light
absorbing substances in the solution
*measures the transmitted light and convert that to the
conc of absorbed light. This conc of absorbed light is
proportional to the conc of your analyte.
*T  A  CONCENTRATION
SINGLE BEAM SPECTROPHOTOMETER
 Simplest type of absorption spectrometer
 One measurement at a time at one specified
wavelength
 The absorption maximum of the analyte must
be known in advance.
*You need to have a information regarding to
the absorbance capacity of your analyte. So that
u can set the spectrophotometer at 450 for ex.
PARTS OF THE SPECTROPHOTOMETER
1. LIGHT/ RADIANT SOURCE
- Provide polychromatic light
-2 types:
Continuum source
=emits radiation that changes in intensity
=widely used in the laboratory
=Tungsten, deuterium and xenon lamps
=Tungsten- commonly used light source in the visible
and near infrared region
=deuterium lamp routinely used to provide UV
=Xenon-UV and visible
Line Source
=emits limited radiation and wavelength
*If continuum is wide wavelength, Line is narrow
=mercury and sodium vapor lamps and hollow cathode
lamp (AAS- Atomic Absorption Spectrophotometry)
2. MONOCHROMATOR
-It isolates specific or individual wavelength of light
*purpose is to select your specific wavelength that is
appropriate for the determination of your analyte
KINDS OF MONOCHROMATOR
 Prisms
 Diffraction gratings
=most commonly used; better resolution than prism
 Filters
=simple, least expensive, not precise but useful
  Holographic gratings
3. CUVET
 It is also called absorption cell/ analytical cell/
sample cell
 Holds the solution
 Alumina silica glass: most commonly used
 Quartz/ plastic: visible and UV
 Borosilicate glass
 Soft glass
 Cuvets with scratches should be discarded
*scratches can cause scattering of light
 Silica cuvettes transmit light effectively at
wavelength is greater than or equal to 220 nm.
 Alkaline solution should not be left standing in
cuvets for prolonged period
*because it can cause corrotion; damage cuvet
4. PHOTODETECTOR
 It detects and converts transmitted light into
photoelectric energy
*the amount of light that was able to pass
through the cuvet is called the transmitted light;
the light that remained or absorbed the cuvet is
called the absorbed light
 It detects the amount of light that passes
through the sample in the cuvet
a. Barrier layer cell/ Photo cell/ Photovoltaic cell
b. Phototube
c. Photomultiplier tube
 Most commonly used detector: measure visible
and UV
 Excellent sensitivity
 Should never be exposed to room light because
it will burn out
d. Photodiode
5. METER OR READ-OUT DEVICE
-It displays output of the detection system
*LS-----M-----C------PD---(to convert and display the
measured amount of transmitted light or
absorbance/transmittance)---METER
BEER’S LAW
-It states that the concentration of the unknown
substance is directly proportional to the absorbed light
and inversely proportional to the amount of transmitted
light.
Conc of the Analyte= Absorbance/ Transmittance
*Conc of analyte is directly proportional to absorbance
ex. Conc inc= Absorbance inc; Conc of analyte is
inversely proportional to transmittance ex. Less conc,
more transmittance
ABSORBANCE
 Is the amount of light absorbed
 Mathematically derived from %T
 2-log%T
PERCENT TRANSMITTANCE
 %T=
 = transmitted thru the sample
 = intensity of light striking the sample
 %T=
DOUBLE-BEAM SPECTROPHOTOMETER
-2 TYPES:
a. Double-beam in space- with 2 photodetectors,
sample and reference beam
b. Double-beam in time – one photodetector;
chopper
A. DOUBLE-BEAM IN SPACE
SINGLE-BEAM: LS----M----C----PD
DOUBLE-BEAM: LS----M----C------PD can measure the
C------PD blank & sample
*You can run both simultaneously
B. DOUBLE BEAM IN SPACE
LS----M----C
CHOPPER PD
C
*The reading of the transmitted light is not
simultaneous because you only have 1 photodetector.
Meaning theres a sequence. The chopper will set the
time from one cuvet to the other. The chopper controls
the entry of transmitted light from your cuvet to the
photodetector. You can run it simultaneously; not entry
B. Flame Emission Photometry
  Atoms of some metals, when given sufficient
heat energy as supplied by a hot flame, become
excited and reemit this energy at wavelength
characteristic for the element.
 Used primarily to determine the concentration
of sodium, potassium, and lithium
 Primary components
Flame – light source and cuvet
Monochromator (3) – 589 nm (sodium) , 767
nm (potassium), and 671 nm (lithium)
Photodetector (3)Light emitted = concentration
 Not applicable for calcium (less easily excited
and present in lower concentration)
 Source of error- fluctuation of light source
 Solution – add internal standard (lithium)
C. Atomic Absorption Spectrophotometry
 Used to measure concentration by detecting
absorption of electromagnetic radiation by
atoms rather than molecules.
 Determination of: Aluminum, Calcium, Copper,
Lead, Magnesium, Lithium, Zinc
 100 times more sensitive than FES
 The amount of light absorbed is proportional to
the concentration
 Ground state – excited state
 The excited atom then returns to ground state,
emitting light of the same energy as it
absorbed.
Parts:
1. Light source:
a. Electrodeless discharge lamp
b. Hollow-cathode lamp
2. Beam Chopper
 Modulates the hollow cathode light beam
 Produce pulses of light
3. Nebulizer
 Delivers a fine spray of sample containing
metallic ion into the flame
4. Atomizer
 Flame atomizer
 Electrothermal atomizer – graphite furnace
(flameless AAS)
 Dissociates the solution into its neutral and
individual atoms
5. Monochromator
6. Photodetector
 Photomultiplier tube – most sensitive, it can
detect quick burst of light
*The purpose of you monochromator and
photodetector is to measure the pulses of emitted light.
The emitted light is directly proportional to the
absorbed light. The amount of absorbance is directly
proportional your concentration of analyte. Meaning,
more emitted light=more absorbed light=higher
concentration.
II. Turbidimetry
 For measuring abundant large particles and
bacterial suspension
 Determines the amount of light blocked by a
particulate matter in a turbid solution
 It depends on specimen concentration and
particle size
 The measurement of reduction of light is due to
particle formation
III. Nephelometry
 Measuring the amount of
_____________________________
 It determines the amount of scattered light by a
particulate matter suspended in a turbid
solution
IV. Electrophoresis
 Is the migration of charged particles in an
electric field
*those particles have different length of
migration
 Purpose: Separation of proteins on the basis of
their electric charge densities
*proteins are amphoteric substances, meaning,
it can contain a positive or a negative charge
depending on the ph. If basic= negative;
acidic=positive
 During electrophoresis, proteins are negatively
charged and they move towards the anode
Types of Electrophoresis  pH and ionic strength of the buffer affects the
analyte
Iontophoresis
 Barbital – 8.6
 Migration of small ions  Tris-boric EDTA – 8.6
 Acidic – cation – migration to the cathode
Zone Electrophoresis
 Basic – Anion – migrate to the anode
 Migration of charged macromolecules n a  Ionic strength – low (faster mobility) High
porous support medium (Slower mobility)
 Ex. DNA, proteins, lipoprotein
Support media
Terms:
 A network of interacting fibers or a polymer
Amphoteric that is solid but traps large amount of solvent in
pores or channel inside
 Substance that can have a negative, zero, or  Must not interact with the analyte
positive charge depending on the condition
Ex. Of Support Media
Anion
Cellulose acetate
 Negatively charge
-Separates serum protein into 5 bands
Cation -Isoelectric focusing
 Positively charge -Separates by molecular size

Factors Affecting the Mobility of particles: Agarose gel

1.Net charge -Purified factor of agar

-Neutral
2. Size and shape -Separates proteins into 10-15 bands
-Separates by electrical charge
3. Strength of the electric field

4. Chemical and physical properties of the medium Polyacrilamide gel

-20 or more fractions, isoenzyme

5. Temperature of operation
determination
Components: Separates on the basis of charge and molecular
size
 Power supply
 Buffer Sample
 Support medium
Detecting system
 Sample
 Detecting system  Densitometer– it measures the absorbance of
stain
Power Supply
NOTES:
 Supplies current or voltage in the system
 Drives the molecules through the support  Electrophoretic mobility is directly proportional
medium to net charge and inversely proportional to
 Driving force molecular size and viscosity of the supporting
medium
Buffer
 A particle without a net charge will not migrate,
 Used to provide ions that carry a current and to it remains in the point of application
maintain the pH at a relatively constant value
  At pH 8.6, the gamma globulins move toward -100x more sensitive than spectrophotometry
the cathode, despite the fact that they are
-More specific, they might be two compounds that
negatively charged – endosmosis
absorbs the same light but will fluoresce different light
 After electrophoresis, the gel is treated with a
mild fixative, such as acetic acid, that -Measures fluorescence
precipitates the proteins at the positions to
which they have migrated. They are stained, PARTS
and the gel is dried and cleared of excess stain. -Light Source -Attenuator

-Primary filter -Sample Holder

Applications of Electrophoresis
-Secondary filter -Photomultiplier tube
 DNA Fractionation
 Isoenzyme determination -Read-out Device
 Protein fractionation LIGHT SOURCE
*5 maj groups of proteins: albumin, alpha 1, alpha -Source of the short wave and high energy light
2,beta, gamma
-Mercury and xenon arc
 Lipid fractionation
-To excite the atoms
*separation of lipoproteins (HDL,LDL,etc)
ATTENUATOR
FLUOROMETRY
-Intensifies the light from the light source
LUMINESCENCE
PRIMARY FILTER
-based on an energy exchange process that occurs when
certain compounds absorb electromagnetic radiation, -Excitation monochromator
become excited, and return to the energy level lower -Selects the wavelength that will best absorbed by the
than or equal to their original level. solution to be measured
TYPES OF LUMINESCENCE SAMPLE HOLDER
A. FLUORESCENCE -Cuvet
-Photoluminescence; the emission is basically
immediate and therefore generally only visible if SECONDARY FILTER
the light source is continuously on
-Emission monochromator

B. PHOSPHORESCENCE
-Photoluminescence; it is characterized when
materials can store the absorbed light energy for
some time and release light later
C. CHEMILUMINESCENCE
-The emission of light is created from a chemical
or electrochemical reaction and not from
absorbed of electromagnetic energy.
MOLECULAR LUMINESCENCE SPECTROSCOPY
(FLUOROMETRY)
-Measures the amount of light emitted by a molecule
after excitation by electromagnetic radiation
-Place on the ring angle from the cuvette to avoid light
from reaching the detector
PHOTOMULTIPLIER TUBE
-Converts light energy to its equivalent electrical energy
-Detects the fluorescence light
N0TE:
-Quenching phenomenon: this phenomenon happens
when the excited state of the molecule loses some of its
energy by interaction to other components of the
reaction system