Review of Analytical Methods Part 1: Spectrophotometry

1
Review of Analytical MethodsPart 1
Spectrophotometry

• Roger L. Bertholf, Ph.D.
• Associate Professor of Pathology
• Chief of Clinical Chemistry Toxicology
• University of Florida Health Science
  Center/Jacksonville

2
Analytical methods used in clinical chemistry

• Spectrophotometry
• Electrochemistry
• Immunochemistry
• Other
• Osmometry
• Chromatography
• Electrophoresis

3
Introduction to spectrophotometry

• Involves interaction of electromagnetic radiation
  with matter
• For laboratory application, typically involves
  radiation in the ultraviolet and visible regions
  of the spectrum.
• Absorbance of electromagnetic radiation is
  quantitative.

4
Electromagnetic radiation
Velocity c
5
Wavelength, frequency, and energy

• E energy
• h Planks constant
• frequency
• c speed of light
• ? wavelength

6
The Electromagnetic Spectrum
7
Visible spectrum
Red-Orange-Yellow-Green-Blue
8
Molecular orbital energies
9
Molecular electronic energy transitions
10
Absorption of EM radiation
11
Manipulation of Beers Law
Hence, 50 transmittance results in an absorbance
of 0.301, and an absorbance of 2.0 corresponds to
1 transmittance
12
Beers Law error in measurement
13
Design of spectrometric methods

• The analyte absorbs at a unique wavelength (not
  very common)
• The analyte reacts with a reagent to produce an
  adduct that absorbs at a unique wavelength (a
  chromophore)
• The analyte is involved in a reaction that
  produces a chromophore

14
Measuring total protein

• All proteins are composed of 20 (or so) amino
  acids.
• There are several analytical methods for
  measuring proteins
• Kjeldahls method (reference)
• Direct photometry
• Folin-Ciocalteu (Lowery) method
• Dye-binding methods (Amido black Coomassie
  Brilliant Blue Silver)
• Precipitation with sulfosalicylic acid or
  trichloracetic acid (TCA)
• Biuret method

15
Kjeldahls method
Specimen
16
Direct photometry
?max 280 nm

• Absorption at 200225 nm can also be used (?max
  for peptide bonds)
• Free Tyr and Trp, uric acid, and bilirubin
  interfere at 280 nm

17
Folin-Ciocalteu (Lowry) method
Reduced form (blue)
Phosphotungstic/phosphomolybdic acid

• Sometimes used in combination with biuret method
• 100 times more sensitive than biuret alone
• Typically requires some purification, due to
  interferences

18
Biuret method

• Sodium potassium tartrate is added to complex and
  stabilize the Cu (cupric) ions
• Iodide is added as an antioxidant

19
Measuring albumin

• Albumin is the most abundant protein in serum
  (40-60 of total protein)
• Albumin is an anionic protein (pI4.0-5.8)
• Enriched in Asp, Glu
• ? Albumin reacts with anionic dyes
• BCG (?max 628 nm), BCP (?max 603 nm)
• Binding of BCG and BCP is not specific, since
  other proteins have Asp and Glu residues
• Reading absorbance within 30 s improves
  specificity

20
Specificity of bromocresol dyes
BCG (pH 4.2)
Albumin
green or purple adduct
BCP (pH 5.2)
Absorbance ?
Time ?
21
Measuring glucose

• Glucose is highly specific for ?-D-Glucose
• The peroxidase step is subject to interferences
  from several endogeneous substances
• Uric acid in urine can produce falsely low
  results
• Potassium ferrocyanide reduces bilirubin
  interference
• About a fourth of clinical laboratories use the
  glucose oxidase method

22
Glucose isomers

• The interconversion of the ? and ? isomers of
  glucose is spontaneous, but slow
• Mutorotase is added to glucose oxidase reagent
  systems to accelerate the interconversion

23
Measuring creatinine

• The reaction of creatinine and alkaline picrate
  was described in 1886 by Max Eduard Jaffe
• Many other compounds also react with picrate

24
Modifications of the Jaffe method

• Fullers Earth (aluminum silicate, Lloyds
  reagent)
• adsorbs creatinine to eliminate protein
  interference
• Acid blanking
• after color development dissociates Janovsky
  complex
• Pre-oxidation
• addition of ferricyanide oxidizes bilirubin
• Kinetic methods

25
Kinetic Jaffe method
Fast-reacting (pyruvate, glucose, ascorbate)
Absorbance (? 520 nm)
Slow-reacting (protein)
creatinine (and ?-keto acids)
0
Time (sec) ?
26
Enzymatic creatinine method

• H2O2 is measured by conventional peroxidase/dye
  methods

27
Enzymatic creatinine method

• H2O2 is measured by conventional peroxidase/dye
  methods

28
Measuring urea (direct method)

• Direct methods measure a chromagen produced
  directly from urea
• Indirect methods measure ammonia, produced from
  urea

29
Measuring urea (indirect method)

• The second step is called the Berthelot reaction
• In the U.S., urea is customarily reported as
  Blood Urea Nitrogen (BUN), even though . . .
• It is not measured in blood (it is measured in
  serum)
• Urea is measured, not nitrogen

30
Conversion of urea/BUN
31
Measuring uric acid

• Tungsten blue absorbs at ? 650-700 nm
• Uricase enzyme catalyzes the same reaction, and
  is more specific
• Absorbance of uric acid at ? ? 585 nm is
  monitored
• Methods based on measurement of H2O2 are common

32
Measuring total calcium

• More than 90 of laboratories use one or the
  other of these methods.
• Specimens are acidified to release Ca from
  protein, but the CPC-Ca complex forms at
  alkaline pH

33
Measuring phosphate
?max 340 nm

• Phosphate in serum occurs in two forms
• H2PO4- and HPO4-2
• Only inorganic phosphate is measured by this
  method. Organic phosphate is primarily
  intracellular.

34
Measuring magnesium

• Formazan dye and Xylidyl blue (Magnon) are also
  used to complex magnesium
• 27Mg neutron activation is the definitive method,
  but atomic absorption is used as a reference
  method

35
Measuring iron

• The specimen is acidified to release iron from
  transferrin and reduce Fe3 to Fe2 (ferrous ion)

36
Measuring bilirubin

• Diazo reaction with bilirubin was first described
  by Erlich in 1883
• Azobilirubin isomers absorb at 600 nm

37
Evolution of the diazo method

• 1916 van den Bergh and Muller discover that
  alcohol accelerates the reaction, and coined the
  terms direct and indirect bilirubin
• 1938 Jendrassik and Grof add caffeine and sodium
  benzoate as accelerators
• Presumably, the caffeine and benzoate displace
  un-conjugated bilirubin from albumin
• The Jendrassik/Grof method was later modified by
  Doumas, and is in common use today

38
Bilirubin sub-forms

• HPLC analysis has demonstrated at least 4
  distinct forms of bilirubin in serum
• ?-bilirubin is the un-conjugated form (27 of
  total bilirubin)
• ?-bilirubin is mono-conjugated with glucuronic
  acid (24)
• ?-bilirubin is di-conjugated with glucuronic acid
  (13)
• ?-bilirubin is irreversibly bound to protein
  (37)
• Only the ?, ?, and ? fractions are soluble in
  water, and therefore correspond to the direct
  fraction
• ?-bilirubin is solubilized by alcohols, and is
  present, along with all of the other sub-forms,
  in the indirect fraction

39
Measuring cholesterol by L-B

• The Liebermann-Burchard method is used by the CDC
  to establish reference materials
• Cholesterol esters are hydrolyzed and extracted
  into hexane prior to the L-B reaction

40
Enzymatic cholesterol methods
Cholesterol esters

• Enzymatic methods are most commonly adapted to
  automated chemistry analyzers
• The reaction is not entirely specific for
  cholesterol, but interferences in serum are
  minimal

41
Measuring HDL cholesterol

• Ultracentrifugation is the most accurate method
• HDL has density 1.063 1.21 g/mL
• Routine methods precipitate Apo-B-100 lipoprotein
  with a polyanion/divalent cation
• Includes VLDL, IDL, Lp(a), LDL, and chylomicrons

• Newer automated methods use a modified form of
  cholesterol esterase, which selectively reacts
  with HDL cholesterol

42
Measuring triglycerides
Triglycerides

• LDL is often estimated based on triglyceride
  concentration, using the Friedwald Equation
• LDL chol Total chol HDL chol
  Triglyceride/5

43
Spectrophotometric methods involving enzymes

• Often, enzymes are used to facilitate a direct
  measurement (cholesterol, triglycerides)
• Enzymes may be used to indirectly measure the
  concentration of a substrate (glucose, uric acid,
  creatinine)
• Some analytical methods are designed to measure
  clinically important enzymes

44
Enzyme kinetics
The Km (Michaelis constant) for an enzyme
reaction is a measure of the affinity of
substrate for the enzyme. Km is a thermodynamic
quantity, and has nothing to do with the rate of
the enzyme-catalyzed reaction.
45
Enzyme kinetics
46
The Michaelis-Menton equation
The Lineweaver-Burk equation is of the form y
ax b, so a plot of 1/v versus 1/S gives a
straight line, from which Km and Vmax can be
derived.
47
The Michaelis-Menton curve
48
The Lineweaver-Burk plot
49
Enzyme inhibition

• Competitive inhibitors compete with the substrate
  for the enzyme active site (Km)
• Non-competitive inhibitors alter the ability of
  the enzyme to convert substrate to product (Vmax)
• Un-competitive inhibitors affect both the enzyme
  substrate complex and conversion of substrate to
  product (both Km and Vmax)

50
M-M analysis of an enzyme inhibitor
51
L-B analysis of an enzyme inhibitor
52
Measuring enzyme-catalyzed reactions

• The progress of an enzyme-catalyzed reaction can
  be followed by measuring
• The disappearance of substrate
• The appearance of product
• The conversion of a cofactor

53
Measuring enzyme-catalyzed reactions

• When the substrate is in excess, the rate of the
  reaction depends on the enzyme activity
• When the enzyme is in excess, the rate of the
  reaction depends on the substrate concentration

54
Enzyme cofactors
Nicotinamide adenine dinucleotide (NAD, oxidized
form)
55
Enzyme cofactors
NADH (reduced form)
56
NAD UV absorption spectra
57
Enzyme reaction profile
58
Measuring glucose by hexokinase

• The hexokinase method is used in about half of
  all clinical laboratories
• Some hexokinase methods use NADP, depending on
  the source of G-6-PD enzyme
• A reference method has been developed for glucose
  based on the hexokinase reaction

59
Measuring bicarbonate

• The specimen is alkalinized to convert all forms
  of CO2 to HCO3-, so the method actually measures
  total CO2
• Enzymatic methods for total CO2 are most common,
  followed by electrode methods

60
Measuring lactate dehydrogenase

• Both P?L and L?P methods are available
• At physiological pH, P?L reaction if favored
• L?P reaction requires pH of 8.8-9.8
• LD (sometimes designated LDH) activity will vary,
  depending on which method is used

61
Measuring creatine kinase (CK)

• Both creatine and phosphocreatine spontaneously
  hydrolyze to creatinine
• The reverse (PCr?Cr) reaction is favorable,
  although the reagents are more expensive
• All methods involve measurement of ATP or ADP

62
Measuring creatine kinase

• Potential sources of interferences include
• Glutathione (Glutathione reductase also uses
  NADPH as a cofactor)
• Adenosine kinase phosphorylates ADP to ATP
  (fluoride ion inhibits AK activity
• Calcium ion may inhibit CK activity, since the
  enzyme is Mg-dependent.

63
Measuring creatine kinase

• Since the forward (Cr ?PCr) reaction is slower,
  the method is not sensitive
• Luminescent methods have been developed, linking
  ATP to luciferin activation

64
Measuring alkaline phosphatase

• The natural substrate for ALKP is not known

65
Measuring transaminase enzymes

• Pyridoxyl-5-phosphate is a required cofactor
• Oxaloacetate and pyruvate are measured with their
  corresponding dehydrogenase enzymes, MD and LD

66
Measuring gamma glutamyl transferase

• Method described by Szasz in 1969, and modified
  by Rosalki and Tarlow

67
Measuring amylase
?(1?4)

• Hydrolysis of both ?(1?4) and ?(1 ?6) linkages
  occur, but at different rates.
• Hence, the amylase activity measured will depend
  on the selected substrate
• There are more approaches to measuring amylase
  than virtually any other common clinical analyte

68
Amyloclastic amylase method
Starch I2

• The rate of disappearance of the blue complex is
  proportional to amylase activity
• Starch also can be measured turbidimetrically
• Starch-based methods for amylase measurement are
  not very common any more

69
Saccharogenic amylase method

• Several methods can be used to quantify the
  reducing sugars liberated from starch
• Somogyi described a saccharogenic amylase method,
  and defined the units of activity in terms of
  reducing equivalents of glucose
• Alternatively, glucose or maltose can be measured
  by conventional enzymatic methods

70
Chromogenic amylase method
Dye-labeled starch

• JJ Vitros application allows small dye-labeled
  fragments to diffuse through a filter layer
• Abbott FP method uses fluorescein-labeled starch

71
Defined-substrate amylase method
4-NP-(Glucose)7
?max 405 nm

• ?-Glucosidase does not react with
  oligosaccharides containing more than 4 glucose
  residues
• A modification of this approach uses
  ?-2-chloro-4-NP, which has a higher molar
  absorptivity than 4-NP

72
Measuring lipase (direct)

• The Cherry/Crandall procedure involves lipase
  degradation of olive oil and measurement of
  liberated fatty acids by titration
• Alternatively, the decrease in turbidity of a
  triglyceride emulsion can be monitored
• For full activity and specificity, addition of
  the coenzyme colipase is required

73
Measuring lipase (indirect)

• Indirect methods for lipase measurement focus on
• Enzymatic phosphorylation (Glycerol kinase) and
  oxidation (L-?-Glycerophosphate oxidase) of
  glycerol, and measurement of liberated H2O2
• Dye-labeled diglyceride that releases a
  chromophore when hydrolyzed by lipase
• Several non-triglyceride substrates have been
  proposed, as well

74
Post-test
Identify the methods proposed by the following

• Folin-Wu
• Jendrassik-Grof
• Somogyi-Nelson
• Kjeldahl
• Lieberman-Bourchard
• Rosalki-Tarlow
• Jaffe
• Bertholet
• Fisk-Subbarrow

• Glucose
• Bilirubin
• Glucose/Amylase
• Total protein
• Cholesterol
• GGT
• Creatinine
• Urea
• Phosphate