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Clinical Chemistry Lab > Lab Exam 2 > Flashcards

Lab Exam 2 Flashcards

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1
Q

What are the laboratory methods for detecting calcium?

A

-redox titrations
-spectrophotometric titrations
-spectrophotometric
-atomic absorption
-ion selective electrodes

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2
Q

What all use atomic absorption as the reference method?

A

-calcium
-magnesium
-potassium
-sodium

*most often just used for Ca and Mg

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3
Q

Old method for calcium that is no longer used.

total calcium is precipitated as calcium ___________.
how is it dissolved?

A

oxalate, acidification

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4
Q

With calcium redox titration, the resulting oxalic acid is titrated against ______________ in a redox reaction and the electrical current is monitored.

A

potassium permanganate

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5
Q

detection of fluorescence emitted from the calcien-calcium complex formation.

A

Calcium: Spectrophotometric Titration:

Calcien-Calcium Fluorescence

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6
Q

Calcium: Spectrophotometric Titration: Calcien-Calcium Fluorescence

the fluorescent complex is titrated with _______ (calcium chelator)

A

EGTA

*volume of added EGTA directly proportional to calcium

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7
Q

what interferences are there with Calcien-Calcium Fluorescence?

A

no interference from magnesium and phosphate

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8
Q

Calcein-Calcium Fluorescence is useful in Stat labs. Research labs routinely use _______, _______, _______ complexes with calcium-binding domains.

A

Flura-2, Fluo3, FRET

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9
Q

Calcium: Spectrophotometric Titration: Calcein-Calcium Fluorescence

the fluorescent complex is titrated with _______ (calcium chelator)

A

EGTA

*volume of added EGTA directly proportional to calcium

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10
Q

What are the two types of Calcium spectrophotometric titration colorimetric methods?

A

-O-Cresolphthalein
-Methylthymol blue

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11
Q

Calcium spectrophotometric titration colorimetric methods:

O-Cresolphthalein method, calcium complexes with o-cresolphthalein in _______________ conditions

A

alkaline (around 12 pH)

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12
Q

What is added for the O-Cresolphthalein method (Calcium) that chelates Mg and increases the specificity?

A

*5-hydroxyquinolone

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13
Q

O-Cresolphthalein calcium method forms a highly colored complex that is measured at ______ nm.

A

580

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14
Q

Calcium: Spectrophotometric titration
colorimetric methods:

Similar principle to O-Cresolphthalein

what is it measured at?

A

Methylthymol Blue

610 nm

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15
Q

Calcium: Spectrophotometric titration
colorimetric methods:

what are other reagents that can be used?

A

alizarin, calcein plasmo-corinth B, GBHA (glyoxaldis-(2-hydroxyanil)

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16
Q

most calcium is in the ionized form. Which method measures TOTAL calcium?

A

Atomic Absorption*

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17
Q

Most accurate method
reference method for calcium

A

Atomic Absorption*

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18
Q

is Atomic Absorption a direct or indirect method?

A

indirect

-sample is diluted
-needs monochromator

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19
Q

Why is lanthanum-Cl used in calcium atomic absorption?

A

to bind interfering substances

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20
Q

Calcium: Atomic Absorption:

Serum diluted into ________________

A

lanthanum-Cl

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21
Q

The most commonly used method for Calcium

A

Calcium: Ion Selective Electrode
Potentiometry

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22
Q

Detects ionized Ca2+ with ion selective electrode (ISE)
! Sensor solvent mediator system
❖ Sensor either an ion exchanger or neutral carrier
➡ selection based on charged and steric factors
❖ Solvent mediator disperses sensor evenly in polyvinyl chloride matrix
❖ Ca2+ binding to sensors develop electric potential across membrane

A

Calcium: Ion Selective Electrode
Potentiometry

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23
Q

Calcium: Ion Selective Electrode
Potentiometry:

Solvent mediator disperses sensor evenly in _______________ matrix
❖ Ca2+ binding to sensors develop electric potential across membrane

A

polyvinyl chloride

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24
Q

What specimen should be used for calcium?

A

Serum, plasma, or whole blood

For whole blood: using Ca2+-titrated heparin

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25
Q

Why can’t Na-Heparin and other coagulants be used for calcium?

A

it will decrease calcium levels

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26
Q

Three things for specimen collection and handling with calcium…

A

-Serum, plasma, or whole blood
-Red blood cells should be immediately separated
-Samples should be “anaerobically” collected

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27
Q

why should calcium Samples be “anaerobically” collected?

A

☺loss of pCO2 will ↑ pH and ↓ free Ca2+

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28
Q

What is the stability of calcium samples?

A
  • Ideally analyzed <1hr after collection, 6 hrs at 4°C
  • Serum stored anaerobically at 4°C- 24 hrs
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29
Q

two Laboratory Methods for Detecting Phosphorous

A

-Molybdenum Blue
-Ammonium phosphomolybdate

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30
Q

What is the most commonly used method for detecting phosphorous?

A

Molybdenum Blue (Reduced form)

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31
Q

Phosphate reacts with molybdenum —–> complex structures
ex, (NH4)3[P(Mo3O10)4]

A

Molybdenum Blue (Reduced form)

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32
Q

Molybdenum Blue (Reduced form): phosphate”

following reduction, _____________________ complex is converted to molybdenum blue (structure unknown) that absorbs at ____nm

A

Aminonaphtholsulfonic (ANS) acid

660 nm

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33
Q

❖ Molybdenum-phosphate complex
▪ reaction is conducted in acid and non-reducing conditions

A

Ammonium Phosphomolybdate (Non-Reduced form)

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34
Q

Ammonium Phosphomolybdate (Non-Reduced form) is measured at …

A

340 nm

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35
Q

Ammonium Phosphomolybdate (Non-Reduced form) advantages?

A

▪ Faster reaction time (Simple and fast)
▪ 3-4x increase in absorbance (Stability)

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36
Q

Ammonium Phosphomolybdate (Non-Reduced form) disadvantages?

A

Acidic conditions will hydrolyze…

▪ glucose phosphate
▪ creatine, phosphate
▪ other organic phosphates

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37
Q

Phosphorous:
Specimen Collection and Handling…

A

-Serum or heparinized plasma, or urine
-RBC should be immediately separated
-Best to collect sample after fasting

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38
Q

why is only heparinized plasma, or urine used for phosphorous?

A

Other coagulants interfere with molybdenum complex formation

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39
Q

Phosphate second highest __________ anion

A

intracellular

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40
Q

**why is it Best to collect phosphorous sample after fasting?

A

☺ Serum levels of PO4 lower after meals (effect of insulin)

41
Q

phosphate has a circadian rhythm. when are levels highest?

A

morning

42
Q

Stability of phosphorus…

A

Serum or plasma: stable for 4 days at RT
7 days at 4 °C
indefinitely at -20 °C
❖ Prolonged storage will increase PO4 at room temperature

43
Q

PTH secreted as prohormone ________aa

A

115

44
Q

After secretion of PTH:

A

115 –> 90 –> 84 —>1-34, 35-84

45
Q

RIA (radioimmunoassay, Axsym) for PTH

A

❖ Antisera detect N-terminal and C-terminal
❖ C-terminal antibody fixed to solid phase-incubated with serum
❖ Followed with enzymatically modified N-terminal antibody detection
▪ Chemiluminescence

46
Q

what method uses chemiluminescence?

A

RIA (radioimmunoassay, Axsym) for PTH

47
Q

where are there PTH receptors?

A

renal tubular cells and osteoblasts

48
Q

____ N terminal part is required for PTH-R activation

A

34

49
Q

____PTH (biologically active PTH)

A

1-84

50
Q

1-84 PTH : non-1-84 PTH = __:__

A

4:1

51
Q

Methods for Detecting Vitamin D?

A

-Non-Selective Competitive Protein Binding Assay
-Radio receptor Binding Assay (RRA)
-Radioimmunoassay (RIA)

52
Q

Most Vit. D assays require…

A

1.Deproteinization or extraction
2.Purification
3.Quantification

53
Q

what is the purpose of the deproteinization step (1) for Vit. D?

A

Free metabolites from DBP

54
Q

What is the purpose of the purification step (2) for Vit. D?

A

Diff in polarities because of # of -OH group
* 1,25(OH)2D: 3 hydroxyl [Calcitriol]
* 25(OH)D: 2 hydroxyl [Calcidiol]
* VitD: 1 hydroxyl

55
Q

What is most commonly used for purification for vit. D?

A

Octadecyl (C18)-Silica: widely used (Silicaminicolume)

56
Q

What is the most abundant form of Vit. D?

A

?

57
Q

Vit. D: Non-selective Competitive Protein Binding Assay (CPBA):

what is used for deproteinization?

A

acetonitrile

58
Q

Radiolabeled calcitriol added to serum (Vit D Binding Protein [VDBP]) in
absence and presence of excess unlabeled calcitriol
! Unbound vitamin D precipitated with charcoal
❖ Retention of radiolabel monitored compared to standard curve

A

Vit. D: Non-selective Competitive Protein Binding Assay

59
Q

Advantage of Vit. D: Non-selective Competitive Protein Binding Assay?

A

Simplicity

60
Q

Disadvantage of Vit. D: Non-selective Competitive Protein Binding Assay?

A

Least specific and least sensitive (4-60 ng/ml)

61
Q

Similar principle to VDBP except that calcitriol receptor expressed in cytosol of
chick gut cells or bovine thymus used to improve specificity

A

Vitamin D: Radioreceptor Binding Assay (RRA)

62
Q

Advantage of Vitamin D: Radioreceptor Binding Assay (RRA)

A

significantly increased Sensitivity (22-63 pg/ml)

63
Q

Disadvantage of Vitamin D: Radioreceptor Binding Assay (RRA)

A

❖ Sensitivity comes at price.
❖ Calcitriol receptor is not very stable and
❖ serum needs to be chromatographically separated

64
Q

Antibody with specificity towards calcitrol

A

Vitamin D: Radioimmunoassay (RIA)

65
Q

Advantage of Vitamin D: Radioimmunoassay (RIA)

A

Stable binding reagent (18-65pg/ml)

66
Q

Disadvantage of Vitamin D: Radioimmunoassay (RIA)

A

❖ Abs display some cross-reactivity between calcitrol and calcidiol,
▪ Serum calcidiol and calcitriol chromatographically separated

67
Q

Laboratory Methods for Detecting Magnesium

A

Titration
! Spectrophotometry
! Ion Selective Electrodes
! Atomic Absorption

68
Q

Detection of fluorescence emitted from calcein-magnesium complex formation
! Fluorescent complex is titrated with EDTA (magnesium chelator)
! Volume of added EDTA directly proportional to [Mg2+] (EGTA added for interference from
Ca2+)

A

Calcein-Magnesium Fluorescence

69
Q

Calcein-Magnesium Fluorescence:

what is used as the magnesium chelator?
what is used for interference from calcium?

A

EDTA, EGTA

70
Q

! CPZ selectively complexes with Mg2+
! EGTA chelates Ca2+. Absorbance ↓ at 550 nm & ↑ at 675 nm.
! Reaction is back titrated with EDTA removed Mg2+ with resultant absorbance charges.
! Volume of EDTA added directly proportional to [Mg2+].

A

Chlorophosphonazo III Spectrophotometry

71
Q

Chlorophosphonazo III Spectrophotometry:

absorbance?

A

decreased at 550 nm, increased at 675 nm

72
Q

Chlorophosphonazo III Spectrophotometry:

Reaction is back titrated with…

A

EDTA removed Mg2+

73
Q

Chlorophosphonazo III Spectrophotometry:

Volume of ______ added directly proportional to [Mg2+].

A

EDTA

74
Q

(most common) Magnesium: Spectrophotometric?

A

Calgamite method

75
Q

! Mg2+ reacted with blue-colored calgamite
! pink magnesium-calgamite complex monitored at 532 nm

A

Calgamite method (Mg)

76
Q

Calgamite Mg method is measured at ___ nm

A

532 nm

77
Q

! Mg2+ reacts with methylthymol blue and
! complex monitored at 510 nm and 600 nm.
! Good correlation with atomic absorption method.

A

Methylthymol Blue

78
Q

Methylthymol Blue: Mg measured at ____ and ____ nm

A

510, 600

79
Q

! Mg2+ reacts with magon (under alkaline conditions) and
! Intensity of color monitored at 520 nm.
! DMA method in our lab uses magon (xylidyl blue 1) to complex with Mg2+ and EGTA and DMSO to
minimize Ca2+ and protein interference, respectively)

A

Magon/xylidyl Blue:

80
Q

Magon/xylidyl Blue for Mg is measured at _____ nm

A

520

81
Q

does magnesium Ion selective electrode measure intracellular or extracellular Mg?

A

both

82
Q

Magnesium:
Specimen Collection and handling…

A

-Serum or plasma (must be separated from red blood cells ASAP), and urine

83
Q

why can’t Citrate, oxalate, and EDTA cannot be used as coagulant for Mg?

A

will chelate Mg2+

84
Q

storage for Mg samples?

A

Separated serum or plasma stable for
▪ 7 days at RT
▪ 7 days at 4 °C
▪ indefinitely at -20 °C

85
Q

What is a more accurate indicator of copper levels?

A

cytochrome oxidase activity in leukocytes

86
Q

two methods for zinc testing

A

-* Atomic Absorption spectrophotometry
* Colorimetric

87
Q
  • Pyridazo-resorcinol is used as chromagen.
  • Guanidine used to remove trace metals from protein
  • Cyanide added to complex all metals except zinc.
A

colorimetric for Zinc

88
Q

disadvantage of colorimetric for Zinc?

A

it can still cross-reacts with other divalent cations. ack of metal specificity

89
Q

colorimetric for Zinc:

What is used as the chromagen?

A

Pyridazo-resorcinol

90
Q

colorimetric for Zinc:

___________ used to remove trace metals from protein

A
  • Guanidine
91
Q

colorimetric for Zinc:

__________ added to complex all metals except zinc.

A

Cyanide

92
Q

Ammonia methods?

A
  • Nesslerization
  • Ion Exchange: “**Berthelot”
  • Ion Selective Electrode
  • Enzymatic
93
Q

based on the release of free NH3 gas from sample after the
ADDITION OF STRONG ALKALI.

NH3 gas is captured in strong acid solution and titrated with alkali or
neutralization.

A

Nesslerization (no longer used)
-for ammonia

94
Q

(Ammonia) Ion Exchange: Berthelot: alkaline solution of ________ and __________.

A

phenol, hypochlorite

95
Q

Ammonia is absorbed into a STRONG CATIONIC ION EXCHANGE RESIN and then
measured using the BERTHELOT COLORIMETRIC REACTION

A

Ion Exchange: Berthelot

96
Q

Ammonia + Berthelot ——>

A

“Blue”

97
Q

BERTHELOT COLORIMETRIC REACTION. cant be….

A

used in automatic system

98
Q

-Sample is alkalized and NH3 is liberated ! semipermeable membrane into a
NH4Cl buffer
-Resulting pH increase is monitored
-Using a [H+] ion selective pH electrode.
—> Change in potential is proportional to [NH3

A

Ion Selective electrode for Ammmonia

Clinical Chemistry Lab (7 decks)

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