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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

H
2
N NH
2
O
Urease
2 NH
4
+
+
OH
OH
-
N
-
O O
Urea Phenol Indophenol

max
= 560 nm
Conversion of urea/BUN
( )
( ) dL L
urea mmol N mg
mmol urea mg
dL mg BUN L mg Urea
L dL
mmol urea mg
urea mmol N mg
L mg urea dL mg BUN
/ 10
/ 28
/ 60
) / ( /
/ 1 . 0
/ 60
/ 28
) / ( /
=
=
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
HN
N
H
N
H
N
O
O
O
-
O
2
H
2
O
2
N
H
H
N
N
H
H
2
N
O
O
O
Phosphotungstic acid Tungsten blue
Uric Acid Allantoin
Measuring phosphate
Phosphate in serum occurs in two forms:
H
2
PO
4
-
and HPO
4
-2
Only inorganic phosphate is measured by
this method. Organic phosphate is primarily
intracellular.
H
3
PO
4
+
(NH
4
)
6
Mo
7
O
24

H
+

(NH
4
)
3
[PO
4
(MoO
3
)
12
]

max
= 340 nm
Molybdenum
blue

max
= 600-700 nm
Red.
Measuring magnesium
Formazan dye and Xylidyl blue (Magnon) are also
used to complex magnesium

27
Mg neutron activation is the definitive method, but
atomic absorption is used as a reference method

N
N
H
3
C
OH
HO
SO
3
-
SO
3
-
CH
3
HO
H
3
C CH
3
H
3
C CH
3
O
N
O
O
-
O
-
O
CH
3
N
O
-
O
O O
-
Calmagite

max
= 530 - 550 nm
Methylthymol blue

max
= 600 nm
Measuring iron
The specimen is acidified to release iron from
transferrin and reduce Fe
3+
to Fe
2+
(ferrous ion)
Transferrin : plasma glycoprotein that transport
dietary iron to the liver, etc
N N N N
SO
3
H
SO
3
Na
Bathophenanthroline Ferrozine
Fe
++

max
= 534 nm
Fe
++

max
= 562 nm
Measuring bilirubin
Bilirubin is the yellow breakdown product of
normal heme-catabolism.
Heme is found in hemoglobin

Diazo reaction with bilirubin was first described by
Erlich in 1883
Azobilirubin isomers absorb at 600 nm
N
H
O
N
H
HO
O
N
H
O
OH
N
H
HO
3
S N N
+
Cl
-
N
H
O
OH
N
H
HO
3
S N N
N
H
O
N
H
HO
O
SO
3
H N N
Diazotized sulfanilic acid
Bilirubin (unconjugated)
Azobilirubin (Isomer II)
Azobilirubin (Isomer I)
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
Bilirubin sub-forms
HPLC analysis has demonstrated at least 4 distinct
forms of bilirubin in serum:
o-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%)
o-bilirubin is irreversibly bound to protein (37%)
Only the |, , and o fractions are soluble in water, and
therefore correspond to the direct fraction
o-bilirubin is solubilized by alcohols, and is present,
along with all of the other sub-forms, in the indirect
fraction
Measuring cholesterol by L-B
The Liebermann-Burchard method is used to
establish reference materials
Cholesterol esters are hydrolyzed and extracted
into hexane prior to the L-B reaction
HO
H
2
SO
4
/HOAc
HOO
2
S
Cholesterol Cholestahexaene sulfonic acid

max
= 620 nm
L-B reagent
Enzymatic cholesterol methods
Enzymatic methods are most commonly adapted to
automated chemistry analyzers
The reaction is not entirely specific for cholesterol,
but interferences in serum are minimal
Cholesterol esters
Cholesterol
Cholesteryl
ester
hydroxylase
Choles-4-en-3-one + H
2
O
2

Cholesterol
oxidase
Quinoneimine dye (
max
~500 nm)
Phenol
4-aminoantipyrine
Peroxidase
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

HDL, IDL, LDL, VLDL HDL + (IDL, LDL, VLDL)(s)
Dextran sulfate
Mg
++

Newer automated methods use a modified form of
cholesterol esterase, which selectively reacts with
HDL cholesterol
Measuring triglycerides
LDL is often estimated based on triglyceride
concentration, using the Friedwald Equation:
[LDL chol] = [Total chol] [HDL chol] [Triglyceride]/5
Triglycerides
Glycerol + FFAs
Lipase
Glycerophosphate + ADP
Glycerokinase
ATP
Dihydroxyacetone + H
2
O
2

Glycerophasphate
oxidase
Peroxidase
Quinoneimine dye (
max
~500 nm)
Post-test
Folin-Wu
Jendrassik-Grof
Somogyi-Nelson
Kjeldahl
Lieberman-Bourchard
Jaffe
Bertholet
Glucose
Bilirubin
Glucose/Amylase
Total protein
Cholesterol
Creatinine
Urea
Identify the methods proposed by the following:
Literature
Review of Analytical Method, Roger L. Bertholf,
Ph.D. , University of Florida Health Science Center