CLIN. CHEM.

26/8,1119-1126 (1980)

Creatinine:A Review
Sheshadri Narayanan1 and Harold 0. Appleton2
S-adenosyl-L-methionine
+ guanidoacetic
acid
Measurement of creatinine has many applications. We
review the determination of urinary creatinine as a valid
methyl transferase
S -adenosyl-L-homocysteine
+ creatine
index of completeness of 24-h urine collection, the clinical
(EC 2.1.1.2)
utility of the determination of creatinine clearance ratios,
and measurement of the ratio of the clearance of specific
In muscle
analytes, such as amylase, to the ratio of clearance of
creatine kinase
creatinine. The chemistry and variables that affect the Jaff#{233} Creatine + ATP
creatine phosphate
+ ADP
(EC 2.7.3.2)
reaction are reviewed, and attempts at improvement of
Creatine phosphate
creatine + inorganic phosphate
specificity are discussed. We also review and assess
techniques other than the Jaff#{233}
reaction for measurement
Creatinine
is present not only in serum and erythrocytes
of creatinine.
-

AdditIonal Keyphrases:

urine
renal clearance
.
24-h
urine collection
.
glomerular function . kidney disease
rate of drug elimination . pancreatitis . amylase/creatinine
clearance ratio #{149} Jaff#{233}
reaction, its mechanism and variables
affecting it - kinetic analysis
other techniques
The history of creatinine dates back to 1847, when Liebig
(1) so named a substance he obtained by heating creatine with
mineral acids. In 1885 Horbaczewski
first synthesized
creatmine (2). His synthesis was confirmed by Paulmann, who in
1894 also furnished
proof that creatine was methylguanidoacetic acid and that creatinine was its internal anhydride
(3).
Bloch and Schoenheimer
(4-6), Borsook and Dubnoff (7,
8), and du Vigneaud and his associates (9, 10) established
that
methionine
could serve as a methyl donor to guanidoacet.ate
in vivo to form creatine. The reactions involved in the metabolism of creatine and creatinine at various sites in the body
are summarized
as follows:
In the kidneys
transamidinase
Arginine

+ glycine

ornithine
(EC 2.1.4.1)

+ guanidoacetic

acid

In the liver
transferase

L-Methionine

+ ATP

S-adenosyl-L-methionine

(EC 2.5.1.6)
+ orthophosphate

+ pyrophosphate

1 Department
of Pathology, New York Medical College-Metropolitan Hospital Center, 1901 First Ave., New York, NY 10029.
2 Department
of Biochemistry,
New York Medical College,
Valhalla, NY 10595.
Received July 5, 1979; accepted Feb. 20, 1980.

but in all bodily secretions,

testinal fluids (11). Being
cerebrospinal
fluid (12).

such as sweat, bile, and gastroinultrafiltrable,

it is present in the

Renal Excretion of Creatinine

In 1929, Rehberg (13) suggested that creatinine was filtered
through glomeruli
and concentrated
in the tubules, being
neither reabsorbed nor augmented by secretion. He based this
suggestion on the fact that the ratio between the concentration
of creatinine in urine and that in serum was larger than known
for any other biologically
excreted substance.
Subsequent
work by Shannon (14) has shown that creatinine
clearance
values do indeed approximate
the clearances
of such exogenous substances as inulin, which are excreted principally by
glomerular
filtration and not reabsorbed
or secreted by the
tubules.
The use of creatinine
coefficients,
which express the
amount (in milligrams per kilogram of body weight) of creatinine excreted in 24 h by a healthy individual,
is based on
the assumption
that the amount of creatinine
in urine is related to body weight (15). Creatinine coefficients
can also be
expressed in terms of nitrogen, as in the creatinine nitrogen
coefficient, for which the creatinine nitrogen value is obtained
by multiplying
milligrams of creatinine per 24 h by 0.372.
Urinary output of creatinine correlates better with muscle
mass than with body weight, as evidenced
by the higher creatinine coefficients
in healthy men than in women (16). An
obese individual would be expected to have lower creatinine
coefficients
than a lean individual
(17).
The urinary output of creatinine
varies little with the
amount or nature of the diet and is independent
of diuresis
(18); physical exercise appears to increase it slightly.
The accuracy of 24-h urine collections is commonly checked
by determining
their creatinine content. Folin (19), who first
used this check, found little day-to-day
variation
in the
amount of creatinine excreted in the urine of a healthy individual. Considerable
literature has since accumulated
on the
subject.

CLINICALCHEMISTRY,Vol. 26, No. 8, 1980

1119

Paterson
(20), in a carefully controlled
study designed to
ensure completeness
of urine collection,
noticed that 24-h
urinary creatinine
excretion
was not sufficiently
constant
to
be used as a check on the accuracy of 24-h urine collection.
Scott and Hurley (21) in their study verified the accuracy
of 24-h urine collections
by noting the rate of radioisotope
excretion
during protein turnover
studies.
Among subjects
chosen for the study were four normal men, a hypercholesterolemic woman, and a woman with nephrotic syndrome. The
24-h urine collections
were made for an average of 17 days,
during which isotope excretion was also studied. Results from
isotope excretion
studies established
completeness
of 24-h
urine collection
with a margin of error of much less than 1%.
Even under these rigidly controlled conditions,
however, there
was considerable
variation
in the amount
of creatinine
excreted in a 24-h period. Variations
within the same subject
occurred from time to time. The coefficient
of variation
(CV)
for creatinine
excretion by the same individual
averaged 10%,
whereas among individuals
it was 29%. On the basis of these
results the authors concluded
that determination
of urinary
creatinine
in an accurately
collected
24-h urine sample provides only a rough estimate
of the completeness
of collection
of urine specimens.
Other investigators
have reported
the CV for creatinine
excretion among individuals
to vary from 5.6 to 22.3% (22) and
from 4.1 to 28.2% (23). Variations
of more than 50% in the
urinary excretion of creatinine were reported in one study (24).
Folin, however, reported
a mean coefficient
of variation
of
6.8% (19). Discrepancies
in the literature
related to the constancy of urinary excretion
of creatinine
may perhaps be due
to the inclusion of subjects whose values are at the extremes
of a wide and varied range (20).
The unreliability
of urinary creatinine
as a valid index of
the completeness
of 24-h urine collection
was also demonstrated in a study that included monitoring
the daily excretion
of creatinine
in normal subjects,
pregnant
women, and patients with mental problems
(25). Other investigators
have
also provided evidence that points to the limitations
of urinary
creatinine
excretion
as a guide to the completeness
of 24-h
urine collections
(26-28).
The cumbersomeness
of assessing
the utility of urinary
creatinine determinations
as an index of completeness
of urine
collection arises from the fact that one needs first to ascertain
the normal excretion of creatinine
for the subject. Because of
the day-to-day
fluctuations
in the same subject, creatinine
excretion
of accurately
collected
24-h urine samples would
have to be measured
for several days to ascertain
the normal
excretion
of creatinine
for the subject. Thus, the determination of urinary creatinine
is not practical in terms of assessing
the completeness
of 24-h urine collection.
Although
creatinine
is not reabsorbed
by the tubules, creatinine clearance
values are not an exact estimate
of the gbmerular filtration
rate, because small amounts
of creatinine
are in fact secreted by the tubules. Consequently,
creatinine
clearance values slightly exceed inulin clearance values. Even
so, a measurement
of clearance of endogenous
creatinine offers
a good estimate
of gbomerular
function
(14). Creatinine
clearance (mL/min)
is calculated
from the following formula:
(concn in urine/concn
in serum)
X urine vol (mL/min)
X
(1.73/body
surface area of the patient),
where 1.73 is the average body surface area in square meters (m2). Body surface
area apparently
correlates
with the size of the kidneys. Expressing
creatinine
clearance
in terms of an average body
surface area permits comparison
of clearance
rates in individuals with different
body surface areas. The relation
between creatinine
clearance
and urea clearance
is apparently
fairly constant when urine output is normal. The ratio of urea
clearance to creatinine
clearance is usually 0.60, but the range
can vary from 0.4 to 1.10 (29).
1120

CLINICAL CHEMISTRY, Vol. 26, No. 8, 1980

Creatinine

Measurement

in

Disease

As early as 1909, it was recognized

(30) that urinary excretion of creatinine
was low in muscle disease, especially
in
muscular
dystrophy.
In renal disease, serum creatinine
values do not increase
significantly
until renal function
has been considerably
impaired. Determination
of creatinine
clearance ratios, however,
may more sensitively
indicate renal impairment
(31) and thus
can be useful for following the effect of a course of treatment
in cases of acute glomerulonephritis,
to identify a primarily
gbomerular
dysfunction,
and to assess overall renal function
(32). However, there still are problems
in regard to the specificity of creatinine
clearance. Thus it is difficult to determine
that impairment
is due solely to glomerular
dysfunction,
because the glomerular
filtration
rate is also decreased
in conditions that contribute
to decreased
renal blood flow.
The diagnostic
usefulness
of creatinine
clearance
values
depends on adherence
to standardized
protocol for collection
of urine and serum specimens.
A catheter
should be used for
patients with decreased
urine volume. A creatinine
clearance
value that is 20 to 40% of normal indicates severe renal disease,
values 40 to 60% of normal suggest moderate
impairment,
and
values between 60 to 80% of normal reflect mild dysfunction
(33). Because
overall drug-elimination
rate constants
and
creatinine clearance are linearly related, one can use creatinine
clearance to adjust drug dosage in patients with renal disease
(34). Clearance
ratios of creatinine
and specific metabolites
have their diagnostic
utility; thus, urinary free il-hydroxycorticosteroid/creatinine
ratios in early-morning
urine samples have been used as an index of adrenal function (35), with
ratios in Cushings syndrome significantly
exceeding those for
normal subjects.
Creatinine
clearance
values decrease
with advancing
age
(36); in one study, average values decreased from 140 mL/min
per 1.73 m2 of body surface, at age 30, to 97 mL/min per 1.73
m2 at age 80 (36). In pregnancy,
because of the considerable
increase in gbomerular
filtration
rate, creatinine
clearance
is
increased over the normally encountered
range by 50% or even
more (37).
Amylase/creatinine
clearance
ratios are useful as an indicator of acute pancreatitis
(38). In one study (38), 16 of 17
patients with acute pancreatitis
had a significantly
increased
amylase/creatinine
clearance
ratio, which remained
significantly above normal longer than did either serum or urinary
amylase activity.
The specificity
of amylase/creatinine
clearance
ratio as a
marker for acute pancreatitis
has been questioned
because it
is increased
in disorders
other than acute pancreatitis-for
example,
acute duodenal
perforation
(39) and chronic renal
insufficiency
(40). Although the mechanism
for the increased
excretion
of amylase in such patients
is unknown,
the high
amounts
of lysozyme (EC 3.2.1.17) some of them excrete in
the urine suggest that the increased ratio may be attributable
to decreased
tubular reabsorption
of amylase (39).
In patients with decreased renal function, it has been shown
by administration
of [14C]creatinine
that a significant fraction
of creatinine
is excreted into the gut, where it is subsequently
metabolized
by bacterial
flora to carbon dioxide and methylamine.
Therefore,
in patients
with decreased
renal function the usefulness
of the measurement
of serum creatinine
as an index of gbomerular
filtration
rate is in question
(41).

Assay of Creatinine
Chemistry

by the JaffO Reaction

of the Reaction

Jaff#{233}
in 1886 observed the red color formed when creatinine
reacted with picric acid in an alkaline medium (42). The reaction bears his name, and it has withstood
the test of time.

H
C-N

C=NH

O2N1CH_N

H-lV
CH3
Fig. 1. Structure of creatinine

Fig. 2. Postulated Janovsky complex for the Jaff#{233}

reaction
(54)

In 1904, Folin utilized the Jaff#{233}

reaction to measure creatinine
in urine (43). Greenwald
was the first to make a systematic
study of the chemistry
of the Jaff#{233}
reaction
(44); he ascribed
the red color to a salt of creatinine,
picric acid, and sodium
hydroxide,
and noted that there were at least two places in the
creatinine
molecule where a shift in a hydrogen
atom could
produce a tautomer
(Figure 1): a lactam-lactim
rearrangement
between positions
3 and 4 or a keto-enol
change between positions 4 and 5.
Dimethyl
creatinine,
in which a lactam-bactim
rearrangement is made impossible
by substitution
of all the hydrogen
atoms attached
to nitrogen, also gives the Jaff#{233}
reaction, thus
ruling out that possibility.
Moreover, neither creatinine oxime,
benzylideneacetyl
creatinine,
nor benzylidene
creatinine,
in
all of which a keto-enol
tautomerism
is impossible,
give the
Jaff#{233}
reaction.
However,
possession
of a structure
allowing
keto-enol
tautomerism
is not in itself sufficient
to allow the
Jaff#{233}
reaction, because dimethylol
creatinine
does not give it.
Greenwald
noted that his compound
could not form a salt with
picric acid because the basic nitrogen
had combined
with a
hydroxymethyl
group. Thus he concluded
a molecule must
also have basic properties
to give the Jaff#{233}
reaction.
Greenwald
and Gross (45) also studied the structural
features of the picric acid molecule. They concluded
that the two
nitro groups in the ortho position in the picric acid molecule,
or even all three nitro groups and the hydrogen
in the meta
position,
were the structural
components
that were responsible for the Jaff#{233}
reaction with creatinine.
They based this
conclusion
on the observation
that such analogs of picric acid
as 2,4-dinitrophenol
or 2,4,6-trinitro-m
-cresol, neither
of
which fulfills the above requirement,
failed to give the Jaff#{233}
reaction with creatinine.
Picric acid was assumed to be in the
o-quinone
form in picrates,
and thus the yellow creatininepicrate would have the same configuration
as trinitro-m-cresob
picrate, except that the latter, which does not give the Jaff#{233}
reaction, has a methyl group instead of a hydrogen atom in the
meta position.
Greenwald
concluded
that the Jaff#{233}
reaction
is a result of several events, including
a keto-enob
transformation in the creatinine
molecule and changes in the picric
acid molecule involving the hydrogen
atoms in the meta positions and possibly all the three nitro groups (46).
Greenwalds
work suggests that the red color formed in the
Jaff#{233}
reaction
is a result of 1:1 and 2:1 complexes
between
creatinine
and picric acid. In 1936, Bollinger
isolated a red
solid from the Jaff#{233}
reaction
mixture
and analyzed
it as a
complex formed from 1 mob of picric acid, 1 mol of creatinine,
and 2 mol of sodium hydroxide
(47). Abe pointed out, however, that picric acid itself reacts with sodium hydroxide
to
yield a picrate ion complex (48).
Several investigators
have provided
evidence,
based on
spectroscopic
and chromatographic
studies, that the red color
formed in the Jaff#{233}
reaction
is not due to picramic acid (49,
50). Depending
on the concentration
of sodium hydroxide
used in the Jaff#{233}
reaction, a first and second complex of picric
acid with hydroxide
ions have been reported (51). The former
prevails at low sodium hydroxide
concentration,
whereas the
second is present if sodium hydroxide
concentrations
exceed
1 mol/L (51). The effect of sodium hydroxide
concentration
on the final product
formed in the Jaff#{233}
reaction
has been

substantiated
by spectroscopic
data (52). Increasing
the hydroxide concentration
shifts the absorption
maxima
of the
product
to longer wavelengths
(53). Studies on the kinetics
of the Jaff#{233}
reaction have shown that the observed first-order
rate constant
is a linear function
of hydroxide
concentration
(52, 54).
Butler (54) has pointed
out that the methylene
group of
creatinine
reacts with picrate anion to form a 1:1 Janovsky
complex, as depicted
in Figure 2 (54).
Because the Jaff#{233}
reaction
product
would be expected
to
give absorbance
peaks for methyl and methylene
protons of
creatinine
and the ring proton of picric acid, proton magnetic
resonance studies have been useful in studying the mechanism
of the Jaff#{233}
reaction (53,55). Results from such studies indicate that a 1:1 reddish-orange
adduct of creatinine
and picric
acid forms first, then is slowly converted
to a stable yellow
compound
according
to first-order
kinetics (53, 54). Polarographic studies demonstrate
that when equal concentrations
of creatinine
and picric acid are maintained,
about 10% of the
final product
is a 2:1 complex
of creatinine
and picrate
(56).
Nuclear magnetic
resonance
studies involving
13C do not
support formation
of a ,Janovsky complex, the spectral data
indicating
that attack at the meta position
of the picrate
molecule by the methylene
group of creatinine
is not involved
(53, 57). Instead, bis-complex
formation
through attack at the
meta positions
of picrate
is suggested
by proton
nuclear
magnetic
resonance
data (53, 58). Such a complex,
possibly
of the Janovsky
type, would result from a meta attack on
picrate by either the methylene
or the enolized carbonyl carbon of creatinine
(53). Our present knowledge
on the mechanism of the Jaff#{233}
reaction
rests on the dependence
of the
hydroxide
concentration
on the reaction
rate under
the
pseudo-first-order
conditions
prevailing
when excess picric
acid is used (50,52). Less absorbance
change is noticed as the
hydroxide
concentration
is increased,
with an accompanying
shift in absorption
maxima to longer wavelengths
(53). A rigid
control of hydroxide
concentration
is a prerequisite
for valid
measurement
of creatinine
by the Jaff#{233}
reaction
(53, 54).

Variables

Involved

in the Jaff#{232}
Reaction

Temperature:
The color developed in the Jaff#{233}
reaction has
been attributed
to the formation
of a red tautomeric
form of
creatinine
at temperatures
lower than 30 #{176}C
and a reaction
time not exceeding
15 mm (59). On longer reaction,
methylguanidine
and presumably
picramate,
a reduction
product of
picrate, are produced,
this effect being pronounced
at temperatures
exceeding 30 #{176}C.
Temperatures
between 15 and 25
#{176}C
apparently
have little effect upon color development,
but
the temperature
at which absorbance
is measured
appears to
influence assay results. The absorbances
of the alkaline picrate
blank and the creatinine-picrate
increase with temperature
increases,
but the magnitude
of the increase is not the same
for both (60); however, the ratio of the absorbance
of a sample
containing
creatinine
to that of a creatinine
standard,
measured at several temperatures,
is constant
(61). Apparently,
the concentration
of alkali in the assay determines
how much
the temperature
affects the Jaff#{233}
reaction
(62).
The effect of temperature
on the absorbance
of an alkaline

CLINICALCHEMISTRY,Vol. 26, No. 8, 1980

1121

picrate solution
depends
on the wavelength
at which measurements
are made (63). Between
475 and 520 nm, an increase in temperature
from 25 to 40#{176}C
increases the absorbance, the magnitude
of the increase becoming steadily more
pronounced
as the wavelength
of measurement
decreases from
520 to 475 nm (63). Thus, the error that could result from
improper
control of temperature
is greater at 490 nm and
neglible at 520 nm. A 1 #{176}C
difference
in temperature
could
produce a 0.7 imol/L
error in the estimated
creatinine
concentration
at 490 nm but 10-fold less at 520 nm. However,
because of the decreased
precision associated
with the lower
molar absorptivity
of the alkaline picrate complex at 520 nm
the use of a wavelength
intermediate
between 490 and 520 nm
has been recommended,
to minimize
temperature-induced
effects (63).
Picric acid: The intensity of the color produced in the Jaff#{233}
reaction
is apparently
independent
of the concentration
of
picric acid used (64), although
at higher concentrations
the
rate of development
of color decreases.
The concentration
of
alkali also influences
the color produced,
the color intensity
being greatest when the alkali concentration
is kept low. Thus
the color produced
in the Jaff#{233}
reaction is inversely proportional to the concentrations
of picric acid and alkali.
The absorption
maximum
for alkaline creatinine-picrate
is approximately
485 nm when measurements
are made
against an alkaline picrate blank. However,
absorbances
of
the alkaline picrate blank are reported
to increase steeply at
wavelengths
below 510 nm. These wavelengths
are also sensitive to slight changes in the picric acid concentration.
Improved specificity
has been claimed for measurements
made
at 520 nm (64).
Protein precipitation:
The extent of analytical
recovery of
creatinine
in protein-free
filtrates
depends on the pH of the
precipitant
(60, 65). At pH <2 (trichboroacetic
acid filtrates)
recovery of creatinine
is quantitative.
However, with tungstic
acid filtrates in the pH range of 3 to 4.5 losses due to adsorption to the protein precipitate
have been reported.
pH: The rate of development
of color in the Jaff#{233}
reaction
is a function
of pH (60). The effect of decreasing
pH on the
color intensity of alkaline creatinine-picrate
differs from that
of the alkaline picrate blank (66).
Interferences:
In addition to homologs and some derivatives
of creatinine,
such as glycocyamidine,
5-methylglycocyamidine, and 5-methylcreatinine,
substances
such as ascorbate,
pyruvate,
acetone, and glucose are among the extensive
list
of compounds
that interfere
with the Jaff#{233}
reaction
(44, 67,
69). Plasma contains
less pseudocreatinine
material than do
erythrocytes
(60, 65, 70).Negligible amounts of Jaff#{233}-positive
noncreatinine
chromogens
are found in urine (60, 65).

Improvement
Reaction

of the Specificity

of the Jaff#{233}

Adsorbents
such as kaolin (71)and Lloyds reagent (60, 65,
72) have been used to improve the specificity
of the Jaff#{233}
reaction. The latter, an aluminum
silicate clay, has found wide
application.
The initial claim to the utility of the Lloyds reagent was that it selectively
adsorbed
creatinine,
leaving the
noncreatinine
chromogens
in solution
(65). However,
it was
subsequently
found that in addition
to creatinine
Lloyds
reagent
adsorbs
several
Jaff#{233}-positive keto acids, indole,
glycocyamidine,
various steroids, and pigments (73).Although
ceric sulfate has been used to remove pyruvate
(68,74),which
is not eliminated
by Loyds reagent, its effectiveness
has been
questioned
(60, 75).
Extraction
with ether reportedly
eliminates
interference
from acetone, acetoacetic
acid, fructose, and glucose (76). A
preliminary
oxidation
step with iodine and treatment
with
1122

CLINICAL CHEMISTRY, Vol. 26, No. 8, 1980

zinc before extraction

is claimed to increase specificity
(76).
Apparently,
treatment
with zinc converts
some interfering
keto acids to the hydroxy form, and clinically used dyes such
as phenolsulfonphthalein
or sulfobromophthalein,
if present,
are reduced to their leuco form. However, the specificity of the
iodine-oxidation/ether-extraction
step and its advantage
over
the Lloyds
reagent
modification
have been questioned
(75).
Pretreatment
with a strong cation-exchange
resin before
performing
the Jaff#{233}
reaction by a continuous-flow
assay has
been utilized (77). Introduction
of a dialysis step with a continuous-flow
procedure
reportedly
improves specificity
(78).
The difference
in behavior
of creatinine-picrate
and noncreatinine
chromogens
subsequent
to acidification
has been
exploited
(75), the difference
in absorbance
obtained
before
and 5 mm after acidification
reportedly
representing
true
creatinine
absorbance.
Apparently,
the color due to the creatinine-picrate
complex fades rapidly, whereas the color resulting from noncreatinine
chromogens
is either stable or
fades slowly (75). This method is satisfactory
for estimating
creatinine
values in the normal range, but is less sensitive at
high creatinine concentrations
(79). To correct this limitation,
the ratios of the tungstic acid filtrate, picric acid, and sodium
hydroxide
can be readjusted
to provide optimum
alkalinity
for color development
(79).
Measurement
of absorbance
at 500 nm with two alkaline
picrate reagents buffered at pH 9.65 and 11.50 also reportedly
gives true creatinine
values. Creatinine-picrate
does not
absorb at pH 9.65, whereas the absorbance
at pH 11.50 is due
to both creatinine
and protein. Thus, without deproteinization, the measurement
of absorbance
of the reaction mixture
at pH 11.50 minus the absorbance
of the pH 9.65 reactant
reflects the true creatinine
value because the other commonly encountered
pseudocreatinine
chromogens
do not react
under the conditions
of the assay (80).
Creatinine
can be assayed by the Jaff#{233}
reaction
without
deproteinization,
by monitoring
initial reaction
rates (55,
81,82). The kinetic assay, however, is not entirely free of interference
from Jaff#{233}-reactivepseudocreatinine
chromogens.
Although some have reported that bilirubin does not interfere
in the kinetic assay (81, 83), others have noted a significant
decrease
in creatinine
values when high concentrations
of
bilirubin are present (84). This effect is apparently
related to
the ability of the reagents to oxidize bilirubin to biliverdin very
rapidly before the initial measurement
(85). Furthermore,
results
based upon a proportional
relationship
between
changes in absorbance
vs changes in time may not always be
valid: in one study, despite the fact that the Jaff#{233}
reaction
follows pseudo-first-order
kinetics,
the rate constants
for
different serum samples were dissimilar (86). Incubation
with
the enzyme creatininase
(EC 3.5.2.10) improves specificity
of
the Jaff#{233}
reaction (87); this procedure,
demonstrated
as early
as 1937 (88), has been successfully
adapted
to a discretesample analysis system (87).
Differential
elution of creatinine
and noncreatinine
chromogens by high-performance
liquid chromatography
on
pellicular
cation-exchange
resins and on-line application
of
the Jaff#{233}
reaction
are approaches
for improving
specificity
(89). Noncreatinine
chromogens
are eluted with a sodium
citrate buffer (pH 4.25) before creatinine.
In other procedures
involving
high-performance
liquid chromatography
the absorbance
of creatinine
is measured
in the ultraviolet
region
(90, 91). In one such procedure,
reported
to be rapid and
specific and to have good precision, creatinine
is separated
by
paired-ion
chromatography
and its absorbance
is measured
at 200 nm (90).In an attempt to develop a definitive creatinine
assay, Lim et al. (91) made a preliminary
separation
on a
cation-exchange
resin, followed by separation
on a reversedphase high-performance
liquid-chromatographic
column and

measured
the absorbance
of creatinine
at 254 nm. The purity
of eluted creatinine
was established
by derivatization
and
subsequent
analysis by gas chromatography
and mass spectrometry.
Separation
with conventional
ion-exchange
resins: Some
procedures
involving
cation-exchange
resins give poor recovery (92-94). Creatinine
is adsorbed
onto a strongly acidic
cation-exchange
resin from an acid solution. Because hydrogen ions interfere with the Jaff#{233}
reaction, the resin is converted
to the sodium form before the creatinine
is eluted. The poor
recovery associated
with these procedures
presumably
is due
to the elution and (or) loss of some creatinine
during conversion of the resin to the sodium form; this loss can be circumvented by using a cation-exchange
resin that is already in the
sodium form (95). Results
obtained
were comparable
with
those obtained
with the Jaff#{233}
procedure
involving the use of
Lloyds reagent.
Separation
of creatinine
by anion-exchange
resin and
subsequent
measurement
in the ultraviolet
are time consuming and offer little advantage
over the Lloyds reagentmodified
Jaff#{233}
reaction
(96-98).

Other Methods for Assay

of Creatinine

Reaction
with 3,5-dinitrobenzoic
acid: Creatinine
reacts
with 3,5-dinitrobenzoic
acid at an alkaline pH to give a purple-rose color (99, 100), but the stability of the color has been
questioned,
and the superiority
of this procedure
over the
Jaff#{233}
reaction
without
modification
to include
the use of
Lloyds reagent is doubtful
(101).
Derivatives
of 3,5-dinitrobenzoic
acid have recently
been
used in two procedures
for assay of serum creatinine.
In one,
creatinine
is reacted with 3,5-dinitrobenzoyl
chloride (102);
in the other, with methyl-3,5-dinitrobenzoate
in a mixture of
dimethyl
sulfoxide,
methanol,
and tetramethyl
ammonium
hydroxide
(103). The latter procedure is reported to have good
precision and correlates
well with alkaline picrate procedures.
Protein precipitation
to yield neutral supernates
reportedly
eliminates
interference
by cephalothin,
and thus increases
specificity
of the 3,5-dinitrobenzoate
procedures
over the
picric acid procedure
(103, 104).
Reaction
with
1,4-naphthoquinone-2-sulfonate:
Measurement
of the reaction
product
of creatinine
and 1,4-naphthoquinone-2-sulfonate
(potassium
salt) has been attempted
(105), but reportedly
gives highly spurious
results
(106, 107).
Sakaguchi
color reaction:
Reaction
of creatinine
with onitrobenzaldehyde
in the presence
of alkali forms oxalylmethylguanidine,
which
is subsequently
converted
to
methylguanidine
by heating after neutralization.
The latter
gives the Sakaguchi
color reaction
for monosubstituted
guanidines
by formation
of a red color with a-naphthol,
thymine, sodium hydroxide,
sodium hypochlorite,
and sodium
thiosulfate
(108). Use of the above assay mixture
for the
Sakaguchi
color reaction
increases
the stability
and the intensity of color formed with creatinine
(109). This reaction
presumably
does not measure
the nonspecific
chromogens
encountered
in the Jaff#{233}
reaction
(109).
Coupled
enzyme assays: An ammonia-selective
electrode
coupled to the enzyme creatinine
deiminase
(EC 3.5.4.21) has
been used to measure
creatinine
(110). This procedure
is
specific for creatinine,
provided that the enzyme used is highly
pure and free from ammonia-generating
and -consuming
activities.
A coupled-enzyme
assay for creatinine
involves creatinine
amidohydrolase
(EC 3.5.2.10), creatine
kinase (EC 2.7.3.2)
pyruvate
kinase (EC 2.7.1.40),
lactate dehydrogenase
(EC
1.1.1.27), NADH, and the change in absorbance
at 340 nm
(111). The standard
curve is reported to be linear to 100 mg/L,

no serum deproteinization
is required,
and the method
is
relatively
specific.
Mass fragmentography:
Recently,
a reference
method involving mass fragmentography
has been proposed
(112). The
procedure
entails adding a known amount
of 5N2-labeled
creatinine
to serum or urine, lyophilization
of this mixture,
and subsequent
isolation of creatinine
by high-performance
liquid chromatography.
The creatinine
is derivatized
and
analyzed
with a gas chromatograph-mass
spectrometer
system. Values obtained
for urinary creatinine
by this method
and the Jaff#{233}
reaction
were almost identical;
with serum,
however, values obtained
by the Jaff#{233}
reaction were considerably higher, suggesting
that the proposed reference method
is more nearly specific for creatinine.

Evaluation of Methodology
Three methods are believed to be the most practical for the
measurement
of creatinine
(109): They are (a) the Jaff#{233}
reaction performed
after treatment
of the sample with Lloyds
reagent (65), (b) the Jaff#{233}
reaction modified to include iodine
oxidation
and ether extraction
(76), and (c) the reaction
of
creatinine
with o -nitrobenzaldehyde
to yield methylguanidine, which then is measured
with the Sakaguchi
color reaction (108).
The specificity
of these procedures
for measurement
of
creatinine
in urine were evaluated
by separately
subjecting
urine and pure creatinine
to a counter current
distribution
between acetate buffer (pH 4.6) and n-butanol
(113). Results
were evaluated
by comparison
with the theoretical
distribution curve for pure creatinine
expected
in such a countercurrent distribution
system.
None of the procedures
evaluated-the
manual Jaff#{233}
reaction; the Jaff#{233}
reaction adapted
for continuous-flow
assay; the Jaff#{233}
reaction modified by use
of Lloyds reagent and (or) ether extraction;
or the Sakaguchi
color reaction
after treatment
with o-nitrobenzaldehydefollowed the theoretical
distribution
curve for pure creatinine,
thus demonstrating
a lack of absolute specificity.
The Sakaguchi color reaction gave the lowest values for apparent
creatinine of all the methods
evaluated.
Interestingly,
whereas
the other methods
evaluated
measured
glycocyamidine
(0.3
mg of apparent
creatinine
per milligram
of glycocyamidine),
the Sakaguchi
color reaction did not measure this compound.
The Lloyds reagent and ether-extraction-modified
Jaff#{233}
reactions did not measure added acetone,
fructose,
L-ascorbic
acid, urea, histidine,
or aspargine,
all of which were measured
by the unmodified
Jaff#{233}
reaction.
Because
urine contains
negligible
Jaff#{233}-positivenoncreatinine
chromogens,
the interferents
with the unmodified
Jaff#{233}
reaction
were not important,
so that it gave the best results for creatinine
of any
of the procedures
investigated.
In an evaluation
of procedures
involving reaction of creatmine with 3,5-dinitrobenzoate,
the efficacies
of the neutral
supernate
modification
and the acidic supernate
modification
have been compared
(104). The neutral supernate
3,5-dinitrobenzoate
procedure
apparently
correlates
perfectly
with
the Jaff#{233}
procedure
in which acidic supernates
are used.
However, the 3,5-dinitrobenzoate
acidic supernate
procedure
gives about 6% higher creatinine
values than the neutral supernate
3,5-dinitrobenzoate
procedure.
This discrepancy
is
related to the analytical
recovery of 99.6% of added creatinine
in the former procedure,
compared with 94% in the latter. Not
surprisingly,
the acidic supernate
3,5-dinitrobenzoate
procedure is less subject to interferences
by acetoacetate,
glucose,
and ascorbate
than is the alkaline
picrate
Jaff#{233}
reaction.
However, the neutral supernate
3,5-dinitrobenzoate
procedure
is subject to much less interference
by cephalothin,
a widely
used antibiotic,
than is the Jaff#{233}
reaction.
Because

of its better
CLINICAL

precision,
CHEMISTRY,

the neutral

supernate

3,5-

Vol. 26, No. 8, 1980

1123

dinitrobenzoate
procedure
is favored over its acidic supernate
modification,
and over procedures
involving modifications
of
the Jaff#{233}
reaction.
It is intriguing,
however,
that the Jaff#{233}
reaction, which emerged before the dawn of this century, still
holds sway, albeit with several modifications,
in the clinical
chemistry
laboratory
as the most widely used technique
for
the routine assay of creatinine.
S. N. is deeply indebted to Dr. Victor Tchertkoff, Professor of Pathology, New York Medical College, and Director of Laboratories,
Department of Pathology, Metropolitan Hospital Center, New York,
N.Y, and to Dr. Joseph G. Fink, Clinical Pathologist, Nassau Hospital,
Mineola, Long Island, NY, for their continuing encouragement,
guidance, and interest during the preparation of this manuscript.

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