Lab Exam 2 Flashcards
What are the laboratory methods for detecting calcium?
-redox titrations
-spectrophotometric titrations
-spectrophotometric
-atomic absorption
-ion selective electrodes
What all use atomic absorption as the reference method?
-calcium
-magnesium
-potassium
-sodium
*most often just used for Ca and Mg
Old method for calcium that is no longer used.
total calcium is precipitated as calcium ___________.
how is it dissolved?
oxalate, acidification
With calcium redox titration, the resulting oxalic acid is titrated against ______________ in a redox reaction and the electrical current is monitored.
potassium permanganate
detection of fluorescence emitted from the calcien-calcium complex formation.
Calcium: Spectrophotometric Titration:
Calcien-Calcium Fluorescence
Calcium: Spectrophotometric Titration: Calcien-Calcium Fluorescence
the fluorescent complex is titrated with _______ (calcium chelator)
EGTA
*volume of added EGTA directly proportional to calcium
what interferences are there with Calcien-Calcium Fluorescence?
no interference from magnesium and phosphate
Calcein-Calcium Fluorescence is useful in Stat labs. Research labs routinely use _______, _______, _______ complexes with calcium-binding domains.
Flura-2, Fluo3, FRET
Calcium: Spectrophotometric Titration: Calcein-Calcium Fluorescence
the fluorescent complex is titrated with _______ (calcium chelator)
EGTA
*volume of added EGTA directly proportional to calcium
What are the two types of Calcium spectrophotometric titration colorimetric methods?
-O-Cresolphthalein
-Methylthymol blue
Calcium spectrophotometric titration colorimetric methods:
O-Cresolphthalein method, calcium complexes with o-cresolphthalein in _______________ conditions
alkaline (around 12 pH)
What is added for the O-Cresolphthalein method (Calcium) that chelates Mg and increases the specificity?
*5-hydroxyquinolone
O-Cresolphthalein calcium method forms a highly colored complex that is measured at ______ nm.
580
Calcium: Spectrophotometric titration
colorimetric methods:
Similar principle to O-Cresolphthalein
what is it measured at?
Methylthymol Blue
610 nm
Calcium: Spectrophotometric titration
colorimetric methods:
what are other reagents that can be used?
alizarin, calcein plasmo-corinth B, GBHA (glyoxaldis-(2-hydroxyanil)
most calcium is in the ionized form. Which method measures TOTAL calcium?
Atomic Absorption*
Most accurate method
reference method for calcium
Atomic Absorption*
is Atomic Absorption a direct or indirect method?
indirect
-sample is diluted
-needs monochromator
Why is lanthanum-Cl used in calcium atomic absorption?
to bind interfering substances
Calcium: Atomic Absorption:
Serum diluted into ________________
lanthanum-Cl
The most commonly used method for Calcium
Calcium: Ion Selective Electrode
Potentiometry
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
Calcium: Ion Selective Electrode
Potentiometry
Calcium: Ion Selective Electrode
Potentiometry:
Solvent mediator disperses sensor evenly in _______________ matrix
❖ Ca2+ binding to sensors develop electric potential across membrane
polyvinyl chloride
What specimen should be used for calcium?
Serum, plasma, or whole blood
For whole blood: using Ca2+-titrated heparin
Why can’t Na-Heparin and other coagulants be used for calcium?
it will decrease calcium levels
Three things for specimen collection and handling with calcium…
-Serum, plasma, or whole blood
-Red blood cells should be immediately separated
-Samples should be “anaerobically” collected
why should calcium Samples be “anaerobically” collected?
☺loss of pCO2 will ↑ pH and ↓ free Ca2+
What is the stability of calcium samples?
- Ideally analyzed <1hr after collection, 6 hrs at 4°C
- Serum stored anaerobically at 4°C- 24 hrs
two Laboratory Methods for Detecting Phosphorous
-Molybdenum Blue
-Ammonium phosphomolybdate
What is the most commonly used method for detecting phosphorous?
Molybdenum Blue (Reduced form)
Phosphate reacts with molybdenum —–> complex structures
ex, (NH4)3[P(Mo3O10)4]
Molybdenum Blue (Reduced form)
Molybdenum Blue (Reduced form): phosphate”
following reduction, _____________________ complex is converted to molybdenum blue (structure unknown) that absorbs at ____nm
Aminonaphtholsulfonic (ANS) acid
660 nm
❖ Molybdenum-phosphate complex
▪ reaction is conducted in acid and non-reducing conditions
Ammonium Phosphomolybdate (Non-Reduced form)
Ammonium Phosphomolybdate (Non-Reduced form) is measured at …
340 nm
Ammonium Phosphomolybdate (Non-Reduced form) advantages?
▪ Faster reaction time (Simple and fast)
▪ 3-4x increase in absorbance (Stability)
Ammonium Phosphomolybdate (Non-Reduced form) disadvantages?
Acidic conditions will hydrolyze…
▪ glucose phosphate
▪ creatine, phosphate
▪ other organic phosphates
Phosphorous:
Specimen Collection and Handling…
-Serum or heparinized plasma, or urine
-RBC should be immediately separated
-Best to collect sample after fasting
why is only heparinized plasma, or urine used for phosphorous?
Other coagulants interfere with molybdenum complex formation
Phosphate second highest __________ anion
intracellular
**why is it Best to collect phosphorous sample after fasting?
☺ Serum levels of PO4 lower after meals (effect of insulin)
phosphate has a circadian rhythm. when are levels highest?
morning
Stability of phosphorus…
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
PTH secreted as prohormone ________aa
115
After secretion of PTH:
115 –> 90 –> 84 —>1-34, 35-84
RIA (radioimmunoassay, Axsym) for PTH
❖ 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
what method uses chemiluminescence?
RIA (radioimmunoassay, Axsym) for PTH
where are there PTH receptors?
renal tubular cells and osteoblasts
____ N terminal part is required for PTH-R activation
34
____PTH (biologically active PTH)
1-84
1-84 PTH : non-1-84 PTH = __:__
4:1
Methods for Detecting Vitamin D?
-Non-Selective Competitive Protein Binding Assay
-Radio receptor Binding Assay (RRA)
-Radioimmunoassay (RIA)
Most Vit. D assays require…
1.Deproteinization or extraction
2.Purification
3.Quantification
what is the purpose of the deproteinization step (1) for Vit. D?
Free metabolites from DBP
What is the purpose of the purification step (2) for Vit. D?
Diff in polarities because of # of -OH group
* 1,25(OH)2D: 3 hydroxyl [Calcitriol]
* 25(OH)D: 2 hydroxyl [Calcidiol]
* VitD: 1 hydroxyl
What is most commonly used for purification for vit. D?
Octadecyl (C18)-Silica: widely used (Silicaminicolume)
What is the most abundant form of Vit. D?
?
Vit. D: Non-selective Competitive Protein Binding Assay (CPBA):
what is used for deproteinization?
acetonitrile
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
Vit. D: Non-selective Competitive Protein Binding Assay
Advantage of Vit. D: Non-selective Competitive Protein Binding Assay?
Simplicity
Disadvantage of Vit. D: Non-selective Competitive Protein Binding Assay?
Least specific and least sensitive (4-60 ng/ml)
Similar principle to VDBP except that calcitriol receptor expressed in cytosol of
chick gut cells or bovine thymus used to improve specificity
Vitamin D: Radioreceptor Binding Assay (RRA)
Advantage of Vitamin D: Radioreceptor Binding Assay (RRA)
significantly increased Sensitivity (22-63 pg/ml)
Disadvantage of Vitamin D: Radioreceptor Binding Assay (RRA)
❖ Sensitivity comes at price.
❖ Calcitriol receptor is not very stable and
❖ serum needs to be chromatographically separated
Antibody with specificity towards calcitrol
Vitamin D: Radioimmunoassay (RIA)
Advantage of Vitamin D: Radioimmunoassay (RIA)
Stable binding reagent (18-65pg/ml)
Disadvantage of Vitamin D: Radioimmunoassay (RIA)
❖ Abs display some cross-reactivity between calcitrol and calcidiol,
▪ Serum calcidiol and calcitriol chromatographically separated
Laboratory Methods for Detecting Magnesium
Titration
! Spectrophotometry
! Ion Selective Electrodes
! Atomic Absorption
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+)
Calcein-Magnesium Fluorescence
Calcein-Magnesium Fluorescence:
what is used as the magnesium chelator?
what is used for interference from calcium?
EDTA, EGTA
! 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+].
Chlorophosphonazo III Spectrophotometry
Chlorophosphonazo III Spectrophotometry:
absorbance?
decreased at 550 nm, increased at 675 nm
Chlorophosphonazo III Spectrophotometry:
Reaction is back titrated with…
EDTA removed Mg2+
Chlorophosphonazo III Spectrophotometry:
Volume of ______ added directly proportional to [Mg2+].
EDTA
(most common) Magnesium: Spectrophotometric?
Calgamite method
! Mg2+ reacted with blue-colored calgamite
! pink magnesium-calgamite complex monitored at 532 nm
Calgamite method (Mg)
Calgamite Mg method is measured at ___ nm
532 nm
! Mg2+ reacts with methylthymol blue and
! complex monitored at 510 nm and 600 nm.
! Good correlation with atomic absorption method.
Methylthymol Blue
Methylthymol Blue: Mg measured at ____ and ____ nm
510, 600
! 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)
Magon/xylidyl Blue:
Magon/xylidyl Blue for Mg is measured at _____ nm
520
does magnesium Ion selective electrode measure intracellular or extracellular Mg?
both
Magnesium:
Specimen Collection and handling…
-Serum or plasma (must be separated from red blood cells ASAP), and urine
why can’t Citrate, oxalate, and EDTA cannot be used as coagulant for Mg?
will chelate Mg2+
storage for Mg samples?
Separated serum or plasma stable for
▪ 7 days at RT
▪ 7 days at 4 °C
▪ indefinitely at -20 °C
What is a more accurate indicator of copper levels?
cytochrome oxidase activity in leukocytes
two methods for zinc testing
-* Atomic Absorption spectrophotometry
* Colorimetric
- Pyridazo-resorcinol is used as chromagen.
- Guanidine used to remove trace metals from protein
- Cyanide added to complex all metals except zinc.
colorimetric for Zinc
disadvantage of colorimetric for Zinc?
it can still cross-reacts with other divalent cations. ack of metal specificity
colorimetric for Zinc:
What is used as the chromagen?
Pyridazo-resorcinol
colorimetric for Zinc:
___________ used to remove trace metals from protein
- Guanidine
colorimetric for Zinc:
__________ added to complex all metals except zinc.
Cyanide
Ammonia methods?
- Nesslerization
- Ion Exchange: “**Berthelot”
- Ion Selective Electrode
- Enzymatic
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.
Nesslerization (no longer used)
-for ammonia
(Ammonia) Ion Exchange: Berthelot: alkaline solution of ________ and __________.
phenol, hypochlorite
Ammonia is absorbed into a STRONG CATIONIC ION EXCHANGE RESIN and then
measured using the BERTHELOT COLORIMETRIC REACTION
Ion Exchange: Berthelot
Ammonia + Berthelot ——>
“Blue”
BERTHELOT COLORIMETRIC REACTION. cant be….
used in automatic system
-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
Ion Selective electrode for Ammmonia
