Lab Exam 2 Flashcards by Sarah King

What are the laboratory methods for detecting calcium?

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

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What all use atomic absorption as the reference method?

-calcium
-magnesium
-potassium
-sodium

*most often just used for Ca and Mg

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Old method for calcium that is no longer used.

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

oxalate, acidification

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

potassium permanganate

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

Calcium: Spectrophotometric Titration:

Calcien-Calcium Fluorescence

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Calcium: Spectrophotometric Titration: Calcien-Calcium Fluorescence

the fluorescent complex is titrated with _______ (calcium chelator)

EGTA

*volume of added EGTA directly proportional to calcium

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what interferences are there with Calcien-Calcium Fluorescence?

no interference from magnesium and phosphate

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

Flura-2, Fluo3, FRET

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Calcium: Spectrophotometric Titration: Calcein-Calcium Fluorescence

the fluorescent complex is titrated with _______ (calcium chelator)

EGTA

*volume of added EGTA directly proportional to calcium

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

-O-Cresolphthalein
-Methylthymol blue

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Calcium spectrophotometric titration colorimetric methods:

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

alkaline (around 12 pH)

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

*5-hydroxyquinolone

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

580

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Calcium: Spectrophotometric titration
colorimetric methods:

Similar principle to O-Cresolphthalein

what is it measured at?

Methylthymol Blue

610 nm

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Calcium: Spectrophotometric titration
colorimetric methods:

what are other reagents that can be used?

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

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

Atomic Absorption*

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Most accurate method
reference method for calcium

Atomic Absorption*

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is Atomic Absorption a direct or indirect method?

indirect

-sample is diluted
-needs monochromator

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Why is lanthanum-Cl used in calcium atomic absorption?

to bind interfering substances

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Calcium: Atomic Absorption:

Serum diluted into ________________

lanthanum-Cl

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The most commonly used method for Calcium

Calcium: Ion Selective Electrode
Potentiometry

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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

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Calcium: Ion Selective Electrode
Potentiometry:

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

polyvinyl chloride

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What specimen should be used for calcium?

Serum, plasma, or whole blood

For whole blood: using Ca2+-titrated heparin

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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

____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