Download Detection of Metabolic Disorders

Detection
Chromatographic
of Metabolic
Procedures
Disorders
and Interpretation
of Results
Helen K. Berry, Carolyn Leonard, Helen Peters,
Mary Granger, and Naree Chunekamrai
A scheme for detection of metaboliElisorders utilizing commercial dip tests, spot
plate tests, and paper chromatographic tests is presented. Specific details are
given for preparation and development of chromatograms
for routine screening
of urine specimens for disorders of amino acid and carbohydrate metabolism.
Specialized tests for confirming positive findings in the screening procedures are
described. The results are interpreted with regard to the variations encountered in
testing normal infants and children, children hospitalized with a variety of diseases,
and mentally retarded children. Examples of specific and generalized aminoacidurias
are given.
\?VITH
RECENT
ADVANCES
in the knowledge
of amino
acid,
protein,
and
carbohydrate
metabolism,
and related
inborn errors
of metabolism,
the
physician
must continue
to rely on the laboratory
to provide
rapid
methods
for recognition
of these diseases
(1). This is most important
in instances
in ‘which the disorder
is associated
with mental retardation
that might be prevented
by early diagnosis
and treatment.
Such common complaints
in infants
as vomiting,
diarrhea,
jaundice,
and failure
to grow may be the first clinical
signs of a metabolic
disorder
which
might be recognized
by simple laboratory
tests. Paper chromatography
is a particularly
efficient, versatile,
and inexpensive
tool in the clinical
laboratory
for investigation
of metabolic
disorders
(2-4).
In this paper
are presented
simple screening
tests combined
with paper
chromatographic
procedures
which permit
rapid recognition
of rare metabolic
disorders.
Additional
procedures
for confirmation
or further
study of
positive
findings are also given. These methods
are applicable
to urine,
blood, and other biologic fluids.
From the Children’s
Hospital
Research
versity
of Cincinnati
College of Medicine,
Supported
Development,
Received
Foundation
Cincinnati,
and the Department
Ohio 45229.
of Pediatrics,
by Grant
HD00324
from the National
Institute
of Child Health
National
Institutes
of Health,
U. S. Public
Health
Service.
for publication
Jan. 18, 1968; accepted
for publication
Mar. 12, 1968.
1033
and
Uni.
Human
1034
BERRY ET AL.
Clinical
Chemistry
Procedures
Collection of Specimens
Freshly
voided urine is placed in a bottle containing
a few crystals
of
thyniol
as Preservative
and refrigerated
as
soon
as possible
after
collection.
A fasting
mornmg
sample
is usually
obtained,
although
a
random
sample IIIaY he used.
Expressionof Results
hi earlier
studies
we reported
urinary
excretions
in ternis of creatinine as a reference
sul)stallce
to take into account in(lividual
differetices
in urine
volume. This
was a useful correction
in comparing
excretion
values from adults.
\Iost of the l)10ce(111lS described
are designed
for
screening
speciniens
front infants
and young children.
it is not feasible
to obtain 24-hr. urine specifliens
for screening
inirposes.
Changes
in muscle mass, on which creatinine
excretion
is dependent,
are so great in infants
and young children
that creatinine
is not useful
as a nieans
of correctioti
for differences
in volume.
Excretion
of
creatiiime
is thus an age-depeiident
variable.
We found that coiicentration l)(r unit volume
of urine was less variable
than ally other factor we
could nicastire
iii a random
urine
specimen.
Fluid intake
of infants
and
voung chihirell
is fairly
uniform
from
day to day. When concentrations
are
reported,
urinary
excretions
are
exj)ressed
lS microgranis
PCI.
milliliter.
Initial Screening
Each urine specimen
is tested with spot tests and commercial
dip
sticks.
Test strips
of Combistif
for pH, protein,
and glucose,
and
Phenistix*
for phenylpyruvic
acid or aspirin,
are dipped
in the urine.
Acetest,*
showing the presence
of ketones, and Galatest,t
indicating
the
presence
of reducing
sugars,
are
used
as described
by the manufacturers.
The urine is also tested with Milloii’s
reagent
for tyrosine
and
parahydroxyphenyl
compounds;
with 2,4-dinitrophenylhydrazine
for
keto acids; and with anthrone
reagent
to show tile presence
of all carbohydrates,
including
nonreducing
sugars.
Cyanicle-rtitroprusside
reagent
is used to detect cystine and homocystine.
Table 1 describes
the preparation of the reagents
and their use in the spot tests.
Solvents and Reagents
Composition
reagents
Ames
Denver
of solvents
for spraying
Company,
Chemical
is listed
are given
Elkhart,
md.
Company,
Denver,
Cob.
in Table
in Table
3.
2, and
preparation
of
vol. 14, No. II,
1968
METABOLIC
Table
Reagent
I. Spot
Preparation
TESTS
0.05
gm.
anthrone
cone. H,S04
2,4-Dinitrophenylhydrazine
0.3%
Millon’s
10 gm. mercury
dissolved
ml. cone.
HNO,
and
reagent
diluted
Cyanide-nitroprusside
7’eat & pontiee
of reagent
Anthrone
(w/v)
+ 25
ml.
in 1 N HC1
with
1035
DISORDERS
22 ml.
reaction
3 drops
urine + 12 drops
anthrone;
mix with glass stirring
rod. Positive:
green to dark
blue
2 drops urine + 2 drops reagent;
let stand 5 mm. & add 2 drops
10% (w/v)
NaOH;
stir with
glass rod. Positive:
reddishbrown which persists
in 11
then
2 drops urine + 2 drops reagent.
Positive:
pink or pink-brown
11,0
10% (w/v) sodium cyanide;
(w/v) sodium nitroprusside
l%5
drops urine + 1 drop sodium
cyanide;
let stand
1 mm.;
add
1 drop
sodium
nitroprus.side.
Positive:
immediate
red-pink
(magenta)
for cystine or
indicates
homocystine;
purple
ketone bodies
Preparation of Chromatograms
No
pretreatment
of urine
specimens
is recommended
other
than
I)reservation
by thymol and refrigeration.
For preliminary
screening,
two one-dimensional
chromatograms
and one two-dimensional
chromatogram
are prepared
from each specimen.
Whatman
No. 1 filter
paper
“for
chromatography”
is used for all chromatograms
unless
otherwise
specified.
Sheets
for one-dimensional
chromatograms
are
cut 11 X 18 in., and for two-dimensional
113/2 X 113/2 in.
The one-dimensional
chromatograms
are prepared
as shown in Fig. 1.
The urine spots are placed 3 cm. apart,
approximately
1 in. from the
bottom
of the page. A quick device for marking
this line is made by
cutting notches 3 cm. apart in a plastic ruler. The width of the ruler is
approximately
1 in. Fourteen
samples
can be placed
on a single
one-dimensional
chromatogram.
IJrine,
50 1., is applied
to spots in
increments
of 5 .J., using a 5-.iJ. self-filling
pipet.* Each spot is allowed
to dry thoroughly
before
another
application
is made.
Drying
is
hastened
by blowing warm air over the paper, using a hair dryer. Lead
pencil only is used to write on the filter paper. Many samples
may be
spotted
at the same time by placing
the chromatograms
in racks. The
*Microchemical
Specialties
Company,
Berkeley,
Calif.
1036
BERRY FT AL.
Clinical
Chemistry
same pipet may be used for all the specimens,
but it must be rinsed
thoroughly
in the next urine specimen.
When the samples
have 1)een
applied,
each sheet is stapled in the form of a cylinder
using only Monel
nonrusting
staples.
The ends
of the staple
are bent
outward
to
strengthen
the cylinder
and to facilitate
their removal.
The sheets are
placed in the solvent overnight.
Convenient
for use are chromatographic
jars 18 in. high and 10 in. in diameter,
covered
with double-strength
glass 12 in. square or 11 in. in diameter.
The chromatogranis
are removed
from the solvent the next morning
and air-dried.
They are then cut into sections to be sprayed
with specific
Table
2.
PREP.RATION
OF SOLVENTS
Solvent
Butanol-acetic
acid-water
(BuAc)
Pyridine-acetone-ammonisim
Isopropanol-formic
Preparation
hydroxide
acid-water
Butanol-pyridine-water
120 ml. si-bitt anol
30 ml. glacial acetic
40 ml. water
(PAA)
(IPF)
ml. pyridine
ml. acetone
ml. cOIt(. ammosimuni
ml. waler
SO ml. n-hutanol
SO ml. pyridine
40 ml. water
hydroxide
(BuEtAm)
160 ml. n-biitaiiol
40 ml. 95% ethanol
40 ml. cone.
Isopropanol-ammonium
Ethyl
hydroxide-water
aeetate-pyridine-water
hydroxide
160 ml. isopropanol
20 ml. formic acid
20 ml. water
(BuPyr)
Butanol-ethanol-ammonitim
100
60
10
40
acid
(INII)
(EtAc)
amniorliltm
120 ml. isopropanol
15 ml. cosic. ammosimum
15 ml. water
hydroxide
hydroxide
120 nil. ethyl acetate
50 ml. pyridine
40 ml. water
Water-isopropanol-ammonium
Benzene-propionic
hydroxide*
acid-waler
200 ml. isopropanol
20 ml. water
10 ml. ammonium
100 ml. henzene
40 ml. propionic
3 ml. water
* For
separation
of 2,4-dinitrophenylhydrazones
of keto
acids.
hydroxide
acid
(15 N)
vol. 14. No. II,
1968
METABOLIC
Table
1037
DISORDERS
3. I’REPARATIoN
OI REAGENTS
Reagent
Niurhydrin
and use
Preparation
(Niri)
2 gm. ninhydrin
(1 ,2,3-triketohydrindine
hydrate)
50 ml. ethanol
(95)%
100 nil, water
850 ml. n-hutanol
Stable
Spray
Isal iii
for 2-4
wk.
at room
chromatogram
and
1 gm. isatin
20 ml. acetic acid
480 ml. ethanol
(95%)
Store in refrigerator.
Stable
Spray
Toiruidiuie
blue
(CSA)
chromatogram
1.2 gm. toluidimie
800 ml. acetone
200 ml. water
Stable at room
Sr ulfamuilic acid
temp.
heat
at
for 5-It)
55_9t)0
for 2-4
wk.
arid heat at 900 for 10 miii.
blue
temp.
4.5 gin. si ulfanil Ic tonI
45 ml. cone. Iuyd rot’lrlorie
100 ml. water
acid
\Varni to dissolve
arid then add 355 nil, water.
described
below. Stable at room temp.
l)iazot
ized srulfariilic
acid
(I )SA
)
2.2 gm. sodium
50 ml. water
Chill sodium
nitrate
for 10 miii.
acid.
arid chill for additional
reagent
is stable
for 2-4
spray reagent,
combine
10% (w/v) potassium
llrouwresol
(p-Auuis)
green
(13C(.)
0.2%
(w/v)
Stable
Spray
for 2-4 wk.
chromatogram
p-anisidiuie
0.2 gm. bromeresol
l”errieyariide-iiitropriisside
at room
in ice ball,
or freezer.
Combine
(‘old solutions
15 miii. The diazolized
days in refrigerator.
For
equal parts of DSA and cold
carbonate;
rise immediately.
in 95% ethanol
in refrigerator.
and heat at I 10-120#{176}
for 8 miii.
green
500 ml. 95% ethanol
Neutralize
with
1 N
changes
to green-blue
Stable
Use as
nitrite
Chill 50 ml. sulfanilic
p-Auijsidiuie
miii.
(sodium
sodium
when
salt
may
hut il
hydroxide
tested
be used)
on
filler
color
paper.
temp.
I gm. sodium
hydroxide
dissolved
iii tO ml. water
1 gm. sodium nitroprusside
dissolved
in 10 ml. water
1 gm. potassium
ferricyanide
dissolved
in 10 ml.
water
Salts
are dissolved
separately
arid then
combined.
Mixture
is diluted
with 90 ml. waler.
After standing
for about 20 mm., initial dark color changes
to pale
yellow aii(l is ready
to rise. Stable
for 2-4 wk. iii
ref rigeral or.
1038
BERRY FT AL.
Table
Clinical
3. (Continued)
Reagent
Dichloroquinonechlorimide
Preparat
(DCC)
Chemistry
on
1 gm. dichloroquinonechlorimide
ethanol
in
100
ml.
95%
Spray lightly but evenly on both sides and allow to dry.
Overspray
wit.h solution
of 0.5%
(w/v)
sodium
tetraborate
in water. DCC solution
is stable for 1-2
wk. in refrigerator.
Borate
solution
is stable
2-4
mo. in refrigerator.
Aniline
phthalate
8.5 gm. phthalic
25 ml. ethanol
50 ml. water
anhydride
(95%)
425 ml. n-butanol
5 ml. aniline
Let stand overnight
in refrigerator
before use.
Stable
for 2-4 wk. in refrigerator.
Spray chromatogram
and heat at 110#{176}
for 10 miii.
Naphthoresorcinol
0.2%
(w/v)
naphthoresorcinol
in 95%
ethanol
8.5% (v/v) ortho phosphoric
acid
Immediately
before use combine
1 volume
a(’id with 5 volumes
iiaphthoresorcinol.
Spray
chromatogram
containing
p-Dimethylaminobenzaldehyde
p-1)imethylamin’ieinuiamaldehvde
lodine-azide
(Pl)AB)
pan
and heat
phosphoric
for 10 miii. at 90#{176}
in oven
of water.
2 gm. p-dimethylaminobenzaldehyde
10 ml. cone, hydrochloric:
acid
Dissolve
before adding
90 ml.
in refrigerator.
water.
Stal)le
1-2
0.5 gm. p-dimethylaminociiinamaldehycle
Dissolve
in 20 ml. cone, hydrochloric
acid. I)iluite
200 ml. with water. Stable 4-6 mo. in refrigeratoi’.
wk.
to
50 ml. 0.1 N iodine (aqueous
soluit lou prepared
r’siuig
potassium
iodide)
50 ml. 95% ethanol
1.5 gm. sodium
azide is dissolved
in above mixture,
Stable
approx.
I wk. iii i’efrigei’ator.
reagents
as indicated
in Fig. 1 and 2. Each sheet is then cut into sections.
The section of Sheet 1 containing
the origin, designated
1 A, is stained
with toluidine
blue for acid mucopolysaccharides.
The strips are dipped
into the reagent,
allowed
to dry 1-2 mm., and washed
in 10% (v/v)
acetic acid. A purple ring at the origin indicates
the presence
of metachromatic
staining
material.
Section
lB is sprayed
with isatin and theii heated.
Proline
gives a
turquoise
spot at R1 0.30. Tyrosine
(Hf
0.34) may interfere
if present
in large amounts.
Other amino acids show pink or purple colors; large
amounts
of glycine produce
a bleached area. Quantitative
determination
Vol. 14, No. II,
1968
METABOLIC
1039
DISORDERS
of proline may be made by determining
570-mg filter. Following
measurement
sprayed
with p-dimethylaminobenzaldehyde
presence
of homocitrulline
(a red color
(purple
color at R, 0.15).
the density
of the spot using a
of proline,
Section
B is over(PDAB)
to indicate
the
at H, 0.22) and hydroxyproline
a
0
U
Fig.
1. Diagram
showing
one-
dimensional
screening
chromatogram cut into sections
for development with selective stains:
toluidiuie
Z
R, .55
blue (CSA),
isatin,
amid ninhydrin
(Nm).
Ninhydrin
section
is oversprayed
(BCO).
with
bromcresol
Each chronnatogram
green
is 11
in. high, and spots are placed 3 cia.
apart
arid 1 in. from bottom
edge.
BuAc,
butanol-acetic
acid-water.
0
RF .05
Ci)
BuAc
Section 1C is sprayed
with ninhydrin
and heated as directed.
Phenylalalline
(blue-gray
spot at H, 0.60) and isoleucine/leucine
(purple
spot
at B, 0.70) are measured
quantitatively
from densitometric
readings
with the 545-m
(blue-green)
filter. $-Aminoisobutyric
acid appears
at
R, 0.50-0.55.
Section
C is oversprayed
with bromcresol
green to reveal
organic
acids which appear
as yellow spots against
a blue-green
background.
Hippuric
acid (R, 0.90) and lactic acid (B, 0.82) may be estimated
by
measuring
the area using a planimeter.*
Pyruvic
acid, when present
in
large amounts,
cannot be distinguished
from lactic acid.
Section
water
*Areas
template
the spot
2A is also
to remove
excess
can be measured
in transparent
to be measured
sprayed
using
dye.
with
1)romcresol
Protein
a Keuffel
arid Esser
does
green
and
not migrate
planiuneter.
washed
in the solvent,
it is also useful
plastic of a series of spots of known areas. These
in successioa
uustil the closest area is found.
with
can
to prepare
be matched
a
over
1040
BERRY FT AL.
Clinical
Chemistry
and a green or blue-green
color at the origin indicates
the presence
of
protein.
Section 2B is sprayed
with p-anisidine
and heated as directed
at 120#{176}.
Sugars
appear
as yellow-to-brown
spots which show bright fluorescence
when viewed under ultraviolet
light (long wavelength,
Chromato-Vue).
U)
R
.55
Fig. 2. One-diuuuenisioiual
screening
indicating
chromatograni
sections
to be
developed
with BCG, p.
anisidine,
and DSA. Ab.
breviatioui
a given
in
Tables
2 curd 3.
d.
R
V
05
V
V
J
VV
‘9
C.)
BuAc
Lactose
appears
at B, 0.10, sucrose at B, 0.18, glucose and galactose
at
B, 0.25, and fructose
at II, 0.30; pentoses
are red spots at II, 0.35. The
reagent
is very sensitive
for carbohydrates,
and normal
traces of glucose can l)e seen in most specimens.
Section 2C is sprayed
with diazotized
sulfanilic
acid (DSA)
to locate
phenolic
acids. p-Hydroxyphenylacetic
acid appears
as a red-purple
spot at B, 0.95. m-Hydroxyphenyl
derivatives,
usually dietary
in origin,
appear
as an orange spot at H, 0.84. The latter substances
are common
ut adults, rare in children. p-Hydroxyphenyllactic
acid appears
as a
red-purple
spot at B, 0.82 and can be distinguished
from the metasubstituted
derivatives
by color. p-Hydroxyphenyllactic
acid is rarely
seen
orange
in urine
spot
from
adults.
o-Hydroxyphenylacetic
acid
appears
as
an
at R, 0.95 and can be distinguished
from the p-hydroxyacid in this solvent
system
only by its color. Salicylic
acid
and salicyluric
acid produce
a yellow spot likewise at B, 0.90-0.95.
One two-dimensional
chromatogram
is prepared
for each urine
sample
using 50 l. of urine, as described
for a one-way
sheet. One
sample is placed on each sheet in a corner hA in. from the left edge and
phenylacetic
Vol. 14, No. II,
METABOLIC DISORDERS
1968
1041
1 in. from the bottom of the sheet, as illustrated
in Fig. 3. The sheet is
stapled
and placed in pyridine-acetone-ammonia
(PAA)
solvent
overnight (Table 2). It is thoroughly
air-dried
for 24 hr. Before the second
solvent run, the staple marks along the PAA side are cut off 1/2 in. from
the edge; this prevents
streaking
caused by metal from the staples.
The
Fig.
3.
preparation
Diagram
for
of
two-di-
mensional
chromatogram
for urinary
amino
acids.
Abbreviations
given
in
Table
2.
2
sheet is then turned
at right
angles,
restapled,
and placed
in isopropanol-formic
acid. The second solvent run can be made during
the
day, and the sheets should be removed
from the solvent
after
about
8 hr. After drying
overnight,
the chromatogram
is sprayed
with lflflhydrin.
The sheet is allowed to dry away from direct sunlight
and then
heated
at 85-90#{176}
for 8 mm. The sheets should be stored
in a freezer
until density
measurements
have been made. The ninhydrin
reaction
fades at room temperature,
but chrornatograms
can be kept at low
temperature
for long periods
without
loss of color.
Quantitation of Amino Acids
Each amino acid can be identified
by its characteristic
position
on
the chromatogram,
as seen in Fig. 4. To make quantitative
measurements, chromatograms
of known amounts
of standard
amino acids are
prepared
and run as described
for urine. Density
readings
are measured on a Photovolt
densitometer.
A 545-ms filter and a 4-mm. circular
opening
is used for amino acids sprayed
with ninhydrin.
A standard
curve is prepared
for each amino acid. The maximum
density
of the
spot is a function
of concentration
in the range 0.5-4.0 mg. and can be
used to prepare
standard
curves. For large spots with density
readings
1042
Clinical Chemistry
BERRY FT AL.
above 0.70, the relation
of area
X density
plotted against
concentration
can be llSe(l to prepare
standard
curves
useful
in the upper
ranges
(3-10 pg.). The amino acid determinations
have standard
errors
of
± 0.2 1.tg. in amounts
below
1-2 j.tg. and ± 0.5 j.tg. for those
values
greater
than 2 jLg.
Sugars
Identification
For identification
of sugars
which are very similar
in structure,
cochromatography
is useful.
This consists
of superimposing
known
sugars
over unknown
reducing
substances
to determine
with which
sugar the poSitioll
of the unknown
coinci(les.
Identification
is indicated
w
Urea Creatinine
Threac,n,
0
ci:
>Tourifle
z
0
w
z
g
ILl
acid
Methyl
()
iz
LU
Gtoon\,jGitcine
z
0
Argin,ne
QGiotonriC
acid
Cystin, icy aleine
E?honoiomin.
phosphor,
S
Origin
> ISOPROPYL ALCOHOL-FORMIC ACID-WATER
Fig.
4. Map
ft,unnid in urine
showing
speciniens.
positions
on two-dimensional
cliromuuatograins
of amino
acids
commonly
Vol. 14, No. II,
1968
METABOLIC
DISORDERS
1043
if the sugar is present
ill more
than trace amounts
and has not been
clearly identified
on the screening
chromatogram.
When large aniounts
are present
(as indicated
by a “medium”
or “large”
Combistix
reaction, black reaction
with Galatest,
or dark-green
to blue-green
reactioit
with anthrone),
the positions
of glucose and galactose
usually
overlap
on any chromatogram.
For clear separation
and identification,
the chromatogram
should be repeated
using a reduced volume of urine. A small
amount
of urine (5-20 l.),
depending
on the amount
of sugar shown
by preliminary
screening
sheets,
is placed
on one position
on two
separate
chromatograms.
One sheet of Whatmnan No. 4 filter paper cut
to 14 X 18 in., and one sheet of Whatman
No. 1 filter paper
cut to
11 X 18 in. are used. One-half
the amount
(2.5-10.0
I.Ll.) is placed
on
positions
adjacent
to the larger
volume
of urine:
urine + gahactose,
urine + glucose,
urine + sucrose,
urine + fructose,
urine + lactose.
The 14-in. chromatogram
(Whatman
No. 4 paper)
is run in butanol:
pyri(ime:water
(BuPyr)
overnight
(Table
2) and sprayed
with panisidine
or aniline
phthalate
(Table
3). The chromatogram
using
Whatman
No. 1 paper
is run overnight
in butanol:acetic
acid:water
(BuAc)
and sprayed
with naphthoresorcinol.
Aniline phthalate
is a general reagent
for all reducing
substances.
Naphthoresorcinal
detects
keto sugars-sucrose
and fructose
(5). After developing,
the unknown
is readily
identified
by determining
with which known
sugar the unknown and standard
appear
as a single spot; double spots will appeal’
in all other instances.
The identification
of glucose
and galactose
is
illustrated
in Fig. 5.
If pentoses
are present
in large amounts
(red spots on screening
chromatogram),
these can be identified
by preparing
chromatograms
as described
above, but adding
ribose, xylose, arabinose,
xyulose,
and
deoxyribose
to the smaller
volumes
of urine.
Ethyl acetate-pyridinewater
(EtAc)
is a useful solvent
for separation
and identification
of
pentoses
(3).
Quantitative
Determination
Quantitative
measurements
of glucose,
galactose,
and lactose
are
made by using BuPyr
as the solvent
and p-anisidine
as the reagent.
Two standard
solutions
are prepared.
Solution
A contains,
per milliliter, 5 mg. glucose
and galactose,
and 10 mg. of lactose.
Solution
B
contains,
per milliliter,
1 mg. glucose
and galactose,
and 2 m’. of
lactOse.
TJrilte, 50 /Ll. or less, is placed oti the SliPPt, ilS described
for
oite-dimensioital
chrontatog’raphy.
On the saute sheet with the urine
specimen
are placed duplicate
spots of 5 and 10 pJ. each of both Solutions A and B. The range covered
is 5, 10, 25, and 50 .mg. glucose and
1044
BERRY FT AL.
Clinical
Chemistry
galactose
and twice these amounts
of lactose.
After
resolution
in the
solvent and spraying,
the concentration
may be determined
by reading
the density
of the spots on a Photovolt
densitometer
(445 mj.t) combined with measurement
of the area of the spots. The outline
can be
Q
Q
Fig.
O
0
5. Separation
identification
of
hycochromatography.
Glu,
glucose;
tose;
BuPyr,
pynidine.water.
10).
Urine
5)
5).
Urine
Urine
+
Glu.
Glu.
Gal,
anud
sugars
galac-
butanol-
Gal.
+
Gal.
Bu Pyr
-
Aniline
marked
more clearly
by viewing
the developed
chromatogram
under
ultraviolet
light (365 mp., long wavelength,
Chromato-Vue).
A standard curve is prepared
by plotting
the product
of density
X area against
concentration.
Fructose
and sucrose
can be determined
quantitatively
using BuAc
as solvent
and naphthoresorcinol
as reagent
(s). The standard
range
is 0.5-10.0
g.
of each sugar.
Quantitative
relation
is obtained
by
plotting
the product
of density
(no filter) X area against
concentration.
Specialized Tests
Histidine
appears
as a double red spot at H, 0.10-0.18
in BuAc. In
this solvent,
imidazole
acetic, imidazole
lactic, and imidazole
pyruvic
acids
appear
at B, 0.36, 0.28 and 0.25, respectively.
The last two
imidazole
derivatives
can be separated
better using isopropanol
:formic
acid :water (IPF)
solvent.
Histidine
can be determined
quantitatively
on chromatograms
51/2
in. high
by resolution
in isopropanol-ammonium
hydroxide-water
(TNH) solvent, a run requiring
2-3 hr. Volumes
of 3 and 10 jLI. of urine
are used. The chromatograms
are sprayed
lightly
Ofl both
sides with
(1lazotized
sul fan ii ic acid-potassillm
ca rl)onate
reagent.
Time standard
raltge is 0.3-2.5 g.
The relation
of area X tlensitv
(545 1flL) plotted
against
concentration
is used to prepare
a standard
curve.
The reaction
of tryptophan
with ninhydrin
is not sensitive,
and
Vol. 14, No. II,
1968
METABOLIC
DISORDERS
1045
tryptophait
is itot effectively
separated
from other amino
acids oit
either
of the one- or two-dimemtsioital
chromatograms
described.
Tryptopliait
niay be separate(l
front other ili(lOle derivatives
ott chromatograms
nui in I Nil using p-dimethylanliltocinnamaldehyde
reagent.
For quantitative
determination,
Whatman
No. 4 paper 14 in. in height
should
be used. The range
of standards
is 0.5-2.5 g.
tryptophan.
Density
of the tryptophan
spot (read at 570 mm) and area should be
used to prepare
a standard
curve. Tryptophan
appears
as a blue spot
at R, 0.30; indole acetic acid is a purple
spot at B, 0.40; indole lactic
acid forms a purple
spot at B, 0.44, coinciding
with urea (pink).
The
density
readings
should
be taken immediately
after the reagent
has
dried, while the background
is still light. On standmg,
the background
becomes deep pink.
Iodine azide reagent
is used to locate cystine
and other sulfur-containing
compounds.
Cystine
appears
at H, 0.03 in BuAc as an area of
rapid
decolorization
against
a brown
background;
homocystine
appean’s at B, 0.18.
Argiiiine
may
be determined
using
ferricvaiuide-nitroprusside
reagent.
On chromatogramns
run in BuAc solvent,
arginine
appears
a pink spot at H, 0.12; creatine
(B, 0.36) and creatinine
(B, 0.33)
as
also
react.
The most characteristic
metabolite
in phenylketonuria,
o-hydroxyphenylacetic
acid, can he determined
by resolving
the urine in butanolethanol-ammonium
hydroxide
(BuEtAm)
and spraying
with dichioroquinonechlorimide
(DCC).
o-Hydroxyphenylacetic
acid appears
as a
dark blue spot at B, 0.65. 1)ensity
readings
using a 570-mp. filter can
be used for quantitative
measurement
in the range of 0.5-2.5 zg. Density
and area should be used if larger
amounts
are present.
A blue-green
spot at B, 0.08 is salicyluric
acid from aspim’in or other o-hydroxybeuzeite derivatives.
Chloride
produces
a bleached
area at B, 0.25, and
indican
produces
a pink spot at B., 0.38. if there is any question
regarding
the presence
of o-hydroxypheitylacetic
acid, the urine may be
extracted
with ethyl acetate,
as described
below for concentration
of
phenolic and indolic acids.
Qualitative Identification of Keto Acids
Urine
specimens
giving
positive
reactions
for keto acids
in the
preliminary
testing
may be examined
further
by chromatography
of
the 2,4-dinitrophenyihydrazine
derivative.
Combine
2.5 ml. urine with
2.5 ml. 0.3% (w/v) 2,4-dinitrophenyihydrazine
in a stoppered
tube. Let
stand 10 mm. Add 10 ml. ethyl acetate
and shake 2 mini. Centrifuge
or
let stand to separate
layers.
Remove ethyl acetate
and place in beaker.
1046
BERRY FT AL.
Clinical
Chemistry
with 10 ml. ethyl acetate.
Combine ethyl acetate extracts
and
dry at room temperature
or under reduced
pressure.
I)issolve
residue
in 0.25 ml. of ethanol-ethyl
acetate
(1:1). Use 10 and 25 s.d. of concemitrate (equivalent
to 0.10 and 0.23 ml urine, respectively)
to prepare
chromatograms.
Standards
should
be prepared
from
solutions
of
pyruvic
acid, pheimylpyruvic
acid, -ketogIutaric
acid, or other
keto
acids,
treated
as described
for urine.
Water-isopropanol-ammonium
hydroxide
is used as solvent. No reagent
is required,
since the derivative
is colored.
However,
the spots absorb
under
ultraviolet
light and the
areas of faint spots may be more clearly located.
Re-extract
Extraction of Urine Specimensfor Measurement of Phenolic and Indolic Acids
Place
a 5-mI. aliquot
of urine in a large test tube (23- to 35-mi.
capacity).
Add conc. HC1 to 1)11 1 (about 5 drops is usually
required).
Add 10 ml. ethyl acetate.
Stopper
and shake 2 miii. Allow layers
to
separate
spontaneously,
or centrifuge
to separate
the layers.
Remove
ethyl acetate
(top layer)
and place in small beaker
or evaporating
dish. Repeat
ethyl acetate
extraction
and comnbiiie with first portion
of ethyl acetate.
Evaporate
to dryness
and take up residue
in 0.5 ml.
of 50% (v/v)
ethyl alcohol.
This effects a tenfold
concentration.
The concentrate
thus prepared
contains
such substances
as hippuric
acid, phenylpyruvic
acid, o-hydroxyphenvlacetic
acid, p-hydroxyphenylacetic acid, p-hydroxyphenyllactic
acid, m-hiydroxyphenyl
derivatives
(usually
dietary
in origin,
particularly
in adults),
salicylic
acid, and
many
metabolites
such as salicyhiric,
3-methoxy-4-hydroxymandelic
acid (VMA) ; homovanillic
acid (HVA),
iiidole acetic acid, indole lactic
acid, xanthurenic
acid, and 3-hydroxyindoleacetic
acid (5-HIAA).
Most
of these can he separated
omi a two-dimensional
chronmatogram
using
INH as the first solvent,
followed
by benzene-propionic
acid-water
as
the second
solvent.
Examination
of the chromatogranl
under
ultraviolet light, before developing,
aids in identification.
Sahicylate
and its
derivatives
are blue-white
fluorescent
spots; xanthurenic
acid and other
indole derivatives
are also fluorescent;
hippuric
acid is an absorbing
spot just above p-hydroxyphenylacetic
acid. Diazotized
sulfanilic
acidsodium
carbonate
will react with phenolic
substances.
Indole
derivatives may be located
on a duplicate
chrornatogram
using
Erhich’s
reagent
(p-dimethylaminobenzaldehyde)
or p-dimethylaminocinnamaldehyde.
Interpretation
The
permit
of Results
spot plate, dip tests, and the one-dimensional
chromatograms
the elimination
from further
study of most of the normal
speci-
Vol. 14. No. II,
1968
mens. Abnormal
malities
which
two-dimensional
ing for amino
positive
tests.
METABOLIC
1047
DISORDERS
reactions
serve to identify
certain
biochemical
abnorcan then be subjected
to intensive
investigation.
The
amino acid chromatogram
serves as a general
screenacid disorders,
as well as providing
confirmation
of
Spot Plate and Dip Tests
The spot tests for sugars
are used to detect abnormalities
hydrate
excretion,
which can he confirmed
by chromatography.
are reactions
of various
sugars
with each reagent.
Combislix
Glaneose
Galactose
Fructose
Lactose
Sucrose
+
Galalest
in carboBelow
Anhrone
+
+
+
+
+
+
+
+
+
if glucose is present,
all three prelinminary
tests for sugar-Combistix,
specific for glucose;
Galatest,
for reducing
sugars;
and Anthrone,
general carbohydrate
reagent-will
be positive.
If galactose,
fructose,
or
lactose
is present,
the Combistix
reaction
will be negative
and the
(lalatest
and Anthrone
reactions
will be positive.
These can be distinguished
on the carbohydrate
screening
sheet. If sucrose
is present,
1)0th Combistix
and Galatest
will show negative
reactions,
and Anthrone
should give a positive
reaction.
the
Phenistix
urine
greenish
produces
a green color with phenylpyruvic
shows a purple
color. Bilirubin
in urine
acid. Aspirin
may produce
in
a
color.
The Acetest
stick appears
to be quite sensitive
for acetone.
Pyruvic
acid also produces
a positive
reaction.
2,4-Dinitrophenylhydrazine
test is positive
in the presence
of keto
acids such as phenylpyruvic
acid; pyruvic
acid; x-ketoglutaric
acid;
keto acids derived
from leucine, valine,
and isoleucine;
diacetic
acid;
and acetone
in large quantities.
The keto acid in a specimen
giving a
positive
reaction
can be identified
by chromatography
of the derivative,
as described
in the action on specialized
tests. -Ketoglutaric
acid was
responsible
for the reaction
of most specimens
in which a positive
test
was obtained.
IMiflon’s reagent
shows a strong
positive
reaction
with tyrosine,
phydroxyphenylacetic,
p-hydroxyphenylpyruvic,
and p-hydroxyphenyllactic acid. This test is useful in rapid testing of specimens
from infants
suspected
of tyrosinosis
or in checking
urine samples
from premature
infants
for ascorbic
acid deficiency.
1048
BERRY FT AL.
Clinical
Chemistry
Screening Chromatograms
Toluidine Blue (CSA)
The test for acid inucopolysaceharides
was miegative in niore than
75% of specimens
tested. A trace reaction-a
faint purple
ring at the
point of application
of the specimen-appeared
in approximately
25%
of specimens.
Specimens
from patients
with Hurler’s
syndrome
show
a dark purple spot, as demonstrated
in Fig. 6.
lsatin-PDAB
Proline
and hydroxyproline
were excreted
as the free amino acids in
approximately
equimolar
amounts
by 95% of infants
under 2 weeks of
age. Amounts
rangedl from 10 to 30 mg./day
(0.1. to 0.25 SM/day).
By
4 months
of age, only 25% of infants
excreted
smaller
quantities
of
proline
and hydroxyproline.
Excretion
by older infants
and children
was less than 1 mg./day,
an amount
which cannot
be detected
on tile
preliminary
screening
chromatograms.
Excretion
of proline
by infants
over 6 months
of age in amounts
oven’ 50 ntg./dav
(100 g./ml.)
is a
good indicator
of pathologic
aminoaciduria.
Children
with renal tubular
defects,
such as Lowe’s disease,
Faticoni
syndrome,
and galactosemia
show elevation
of prolimie as an early and
characteristic
feature
of their aminoaciduria.
infants
with ascorbic
acid
deficiency
excrete
increased
amounts
of proline
and hyciroxyproline.
Citrulline
excretion
occurs in Hai’tnUI) disease
and Lowe’s disease.
It
appears
as a deep-purple
spot at B, 0.15. Following
overspray
with
PDAB,
citrulline
is yellow, similar
to urea (B, 0.50). Homocitrulline
(R, 0.22) is found in specimens
from
infants
undem’ 6-8 months
of age
it is rarely
found ut specimens
from oldier children.
(ilycine
in large
amounts,
such as fouiid
in specimens
from
patients
with
hyperglycinuria,
shows a characteristic
bleached
area on the isatin
sheet
(B, 0.22). Cystine,
in specimens
from cystinuric
patients,
forms a deepblue spot at B, 0.10. Other amino acids react with isatin to produce
pink,
gray, or blue colors. Generalized
amntoaciduria
may be suspected
from
examination
of the isatin screening
sheet.
Ninhydrin
(Nm)
Excretion
of phenylalanine
in amounts
above
above 50 /Lg./ml. are indications
of generalized
,,.,
Fig. 6. Screening
Strip
shows
a series
ehromatograms
of specimens
ads
stained
with
from a patient
Pt’,’ry
65 g./nil.
and
aminoaciduria.
#{149}
G
toluidine
blue for acid
with Hurler’s
syndrome.
- bt
leucine
Excre-
0
nnueopolysaccinariules,
Vol. 14. No. II,
1968
METABOLIC
DISORDERS
1049
tion of pheitylalanine
in aniouitts
greater
than 100 g./ml.
with normal
or low excretion
of leucine
suggests
phenylketonuria.
Derivatives
of
penicillin
amtd conjugates
of sahicylic acid produce purple spots with nmhydrin
which ntigrate
ahead
of leucine.
A derivative
of ampicillin
migrates
to R, 0.62 and may be mistaken
for phenylalanine.
The reaction
with ninhdyrin
is purple rather than blue ; care must be taken, however,
in examining
specimens
from children
under therapy.
A yellow spot at
the same position
of phenylalanille
has been seen in specimens
from
infants
with ascorbic
acid deficiency;
this may likewise
interfere
with
measurement
of phenylalanine.
Bromcresol Green (BCG)
over Ninhydrin
Traces
of hippuric
alld
lactic acid ai’e present
in most specimens.
Salicylic
and salicvluric
acids migrate
with hippuric
acid. Phenolic
acids derived
from tyrosine,
tryptophan,
and phenylalanine
also contribute to the acid areas. If the area at R., 0.80 is more than 0.60 sq. in.,
lacticaciduria
or lactic-pyruvicaciduria
should
be suspected.
Pyruvic
acid may be identified
as described
in the section on keto acids.
Sromcresol Green (BCG)
Protein,
as indicated
by the chromatographic
screening
tests, shows
a good corm’elation with dip tests for albumin.
This strip is particularly
useful when specimens
dried on filter paper are tested and when there
has been no opportunity
to carry
out conventional
tests. Protein
in
amounts
over 1000 mg./l00
ml. interferes
with migration
of other substances,
aiid tite chromtogram
is distorted.
p-Anisidine
(p-Anis)
Traces
of lactose,
sucrose,
and liexose
are common.
Excretion
of
lactose
or sucrose
in amounts
above 100 mg./100
ml. should be considered
abliormal,
and disaccharide
intolerance
should be suspected.
If
the area occupied
by the hexose spot is greater
than 0.60 sq. in., the
hexose should be separated
in BuPyr solvent, as described
for identification and quantitative
determination.
Glucose excretion
may be elevated
in diabetes,
renal glycosuria,
and in certain
types
of renal tubular
defects.
Galactose
excretion
is usually
characteristic
of galactosemia,
although
infants
with liver disease
may excrete
small amounts
of this
sugar.
Diazotized Sulfanilic Acid (DSA)
Specimens
from infants
and children
normally
show traces
of phydroxyphenylacetic
acid. if tile phenylalanine
concentration
was
greater
than 100 g./ml.,
the DSA screening
chromatogram
should be
examined
carefully
for an orange
spot of o-hydroxyphenylacetic
acid
at B, 0.90. The o- and p-hydroxyphenylacetic
acids can be separated
S
11
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Vol. 14, No, II,
968
METABOLIC
DISORDERS
1051
using BuEtAm
solvent.
The identity
of o-hydroxyphenylacetic
acid
should be further
confirmed,
and tile amnoutit measured
quantitatively
using DCC reagent.
p-Hydroxyphenyllactic
acid in amounts
greater
than 100 JLg./ml. is an indication
of ascorbic
acid deficiency
in infants.
Of approximately
2000 normal
infants
tested at age 4-6 weeks, 2.1%
excreted
p-hydroxyphenyllactic
acid iii amounts
ranging
from 100 to
2000 g./ml.
This substance
is also found
in urine specimens
from
infants
with untreated
galactosemia
and from infants
with hereditary
tyrosinosis.
When
p-hydroxyphenyllactic
acid is found
on the DSA
screening
strip,
differential
diagnosis
may be made
on the basis of
other
biochemical
and clinical
findings.
Usually
the excretion
of phydroxyphenyllactic
acid is the only biochemical
abnormality
noted in
urine of infants
with ascorbic
acid deficiency.
Specimens
from infants
with tyrosinosis
usually
show a gross
generalized
aminoaciduria.
Glucose,
galactose,
fructose,
or mixtures
of these
sugars
may be
excreted
in tyrosinosis.
Metabolites
of epinephrine
and norepinephrine,
1/MA and HVA,
found in specimens
from patients
with neui’oblastoma,
may be detected
on the DSA screening
strip
(1/MA, red-orange,
B, 0.72; IIVA,
redpurple,
R, 0.88).
Tables
4 and 5 show reactions
in the screening
tests on specimens
from patients
with some of the more common metabolic
disorders.
Tile
pattern
of results
in the preliminary
tests can often aid in recognizing
significant
metabolic
disorders
before the appearance
of clinical symptoms and is useful in selecting
the technics
to he used in confirmatory
tests.
Millon’s
test for tyrosine
and tyrosine
derivatives
was positive
in
specimens
from a premature
infant
with ascorbic
acid deficiency,
a
galactosemic
infant,
and an infaiit with tym’osinosis.
These specimens
were also similar
in having
positive
reactions
with 2,4-dinitrophenylhydrazimie
and with Phenistix.
The keto acid was identified
as phydroxyphenylpyruvic
acid in each instance.
Specimens
from premature infants
usually
show negative
tests for sugars
and for protein;
amino acid excretions
are in the normal range or only slightly
elevated.
Specimens
from patients
with galactoseniia
and tyrosinosis
may have
similar biochemicai
abnormalities,
and the diagnosis
can he established
only by measurement
of galactose-1-yhosphate
uridyl transferase
activity iii ei’vthrocvtes.
Patients
with gellel’ahized
aminoaei(luria-e.g.,
love ‘s disease, Tlartnuii
disease, and Falteolti
svlidi’Oille-SltOwed
posttive reactions
with Milloll ‘s reagent
as a result of increase(l
exeret ion
of tyrosine;
p-hydroxyphenyllactic
acid was not excreted
by these patients.
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5)
55n5?.,,
z.
5s.O5)
,,.,Ca).5)
5C5)5)-,.’
-
-
-;
;;-
;‘
c
Vol. 14, No. II,
The most
1968
METABOLIC
characteristic
1053
DISORDERS
features
of the screeniltg
tests in umiimiespeciare the positive
reactioits
for keto
and Phenistix,
the iuuci’eased
excretion
of pheulylalalliute
together
with low or normal
excretion
of
leucine amid other amino acids, ahl(l the presence
of o-hydroxyphenylacetic acid. Patiemits with Tiartnump disease,
Fanconi
syndirome,
tyrosinosis, and other
types
of generalized
aminoaciduria
also excreted
increased
amounts
of phenylalanine,
but ill these instances
the phenylaianine
was associated
with elevation
of leucine
excretion.
Mixtures
of sugars
are often
excreted
in abnormal
amounts
by patients with tyrosinosis
; in patients
with I we ‘s disease
and FanconiLigrtac
syndrome,
glucose
was time only urinary
sugar
found
iii increased
amounts.
The most
characteristic
abnormality
in urine
from patients
with
Lowe’s disease
was the marked
increase
in excretion
of proline
and
hydroxyproline,
persisting
beyond
infancy.
Elevation
of these
two
amino acids was seen ill specimens
from patients
with galactosemia,
incus from phellylketolluric
Initielits
acid with 1)0th 2,4-diltitrophellylhydraziule
tyrosinosis,
tubular
an(l
Fanconi
syndrome,
reflecting
tile
generalized
renal
defect.
patients
with
Fariconi-Lignac
syndrome,
with
homocvstinuria,
gave positive
reactions
with the
reagent.
it may be worthwhile
to note that cystine
excretion
was not prominent
ill the specimen
from the patient
with
acquired
Fanconi
syndrome.
No other characteristic
al)normahties
were
seen in specimens
from J)atieflts
with cystilniria
or homocystinuria.
Two-dimensional
chromatography
was necessary
to distinguish
the two
disorders.
The significance
of sucrose
excretion
by the homocystinuric
patient
is unknown.
No abnormalities
were revealed
by scmeeluillg tests on specimens
fronu
patients
with hyperglycinemia
other thaI! a large l)leached
area on the
isatin
chromatogram,
characteristic
of glycine.
Particularly,
the
screening
tests indicate
the absence
of ketones
and acidic metabolites.
The specimen
from the patient
with histidinemia
gave positive
tests
Specimens
from
cystinuria
and with
cyanide-nitroprusside
for keto acids,
Two-dimensional
using
both
2,4-diluitropheluylhydrazine
chromatography
and
separation
of
and
the
derivatives
was necessary
for confirmation
of the abnormal
excretion.
In the specimen
from the patient
with Hurler’s
syndrome,
test
for
acid
mucopolysacchai’ides
was
Phenistix.
imidazole
histidine
only
the
positive.
Specimens
from a patient
with pyruvic-lacticaciduria
and from tile
patient with acquired
Fancoumi syndrome
gave strong positive
reactions
with 2,4-dinitrophenyihydrazine,
but not with Phenistix.
The increased
1054
BERRY
FT
Clinical
AL.
Chemistry
o
#{149}
-
.
(-..
-
1
2
3
4
5
6
-
--
7
.4
8
Fig. 7. Chromatogram
of 2,4-dinitroplnenytlnydnazones
of keto acids:
1, a-ketoglutaric
acid;
pyruvic
acid, showinig separation
of lactone
derivative;
3, phenylpyruvic
acid; 4, urine from
patient
suspected
of excreting
pyrntvic
acid;
5, urilne from same patient
with added
pyruvic
,
acid;
with
acid.
6, urine fronn same patient
with a-ketogiutaric
positive
screensinng test for keto acids;
8, same
Note that pyruvic
acid fornns a double spot in
,
acid added;
7, urine from normal
child
urine as in 7 with added
a-ketoglutaric
4, and 5.
vol.
14, No. II,
1968
Table
6.
METABOLIC
AMINo
Acm
ExcRETIoNs
ay
1055
DISORDERS
2250 SURJECTS-BIRTnI
Excreiion
.4
lliptidinie
Glycirie
Serine
Alanninie
Glmntamimte
Threoniiue
Lysine
Tainrinie
1-Aminoisobutyric
(Jiutamic
acid
Tyrosine
Valinie
mists
,lr1,l
lies,,
3S
I1 t,laInIiilf It’
I toot’tlt lIIIIIII(’
FI$, 8 T wn ii nnstsitluou I
t’Iittiititi tttIit#nt
of
IIIItItItnnIIiitI no’ltld slitiwltig
noriiuil
pit It t’ttn l’elne,
1 utntitlnt’,
niloiltin’, lnlIln1lun’1 n’tlne
Alt
tiitnaiy
MIOlUn ,o’kl eInrt’
unit tsintnit
welt’
pin-pa rel
o l tIIIIIC lUlled
tnt lnt’lwlde lutil In-intn’tl
(pg/mi)
±Stnndurd
129
93
60
50
42
acid
TO iS YEARS
36
32
21
19
21
13
10
deriution
132
70
.) i
49
44
30
48
50
33
26
22
23
23
II
M
J0
1056
BERRY
tXci’eti0Ii
of acidic sul)slalices
grain.
Isolation
of the keto acid
\Vits
FT
Clinical
AL.
Chemistry
evitlc’iit on the screellilig
chromatoderivative
was tecessary
to confirm
tile
trence
of pyruvic
acid ( t’ig. 7). JiartIlup
(hisease should be suspected
when plieltylalaltilie
am! leucine
are elevated
withont
elevation
of
proline,
and when tests for sugar,
protein,
all(l phenolic
acids are
negative.
The presence
of abnormal
amounts
of tryptophan
and indole
acids serve to confirm the diagnosis.
Amino Acid Screening
Figure
4 shows a map of amino acids which are commonly
urine
specimeuts.
Those
present
in most specimens
are shown
found
as shaded
#{149}“j?
I,I
/
Fig. 9. Urinary
of hypenglyciuemia
CINCINNATI
amino acid chromatogram
and hypergiycinuria.
showing
marked
excretion
2.
OHIO
of gtycine,
characteristic
in
v0l.
14, No. II,
1968
METABOLIC
1057
DISORDERS
areas, while time outlined
areas represent
extremes
which may be cmicountered.
Positions
of uncommon
amino acids associated
with various
disorders
of amino acid metabolism
are shovti in photographs
of chromatograms
diescribed
below. 1 )ata 01! amino acid excretions
from 2230
Fig.
10.
[Tnimnary
annino
aci(I
grain
showing
cystine.
kinidly
chromato-
honnno-
(Specimen
furnnished
1)r.
Grant
Philadelphia
by
Morrow,
.
)
F.
.
pp
II
children
from birth to 18 years are in Table 6. Results are expressed
as
micrograms
of amino acid per milliliter
of urine.
Figure
8 shows the normal
pattern
of amino acids, characterized
by
the presence
of histidine,
glycine,
serine,
glutamine,
and alanine.
The
concentrations
of these amino acids in a given specimen
are usually
highly correlated.
Traces to moderate
aniounts
of other amino acidslysine, threoiiine,
glutamic
acid, $-aminoisobutyric
acid-may
occur in
normal
specimens.
Leucine, vahine, phenylahaiiine,
tyrosiile,
cystine,
and
methiolhine
are foulid
iii
trace
amounts
only ill normal
specimens.
llomocitrimlhine,
I)roline,
and Imydroxyproline
univ he prominent
amino
acids 011 chromatograms
from young infants.
Iethylhistidine
is a normal constituent
of urine from older children
amid adults.
1058
BERRY
ET
Clinical
AL.
Chemistry
Examples
Fig. 9-18.
of some of the more common aminoacidurias
are shown in
All chromatograms
were
prepared
using
50 l. of urine
otherwise
stated.
The specific aminoacidurias
shown-glycinuria,
unless
homocystinuria,
argininosuccimmicaciduria,
and
citruhlinuria-are
gen-
erally accompaitied
by elevation
of the same amino acid in the blood.
in the generalized
aminoacidurias
shown, it is helpful
in interpreting
the pattern
to determine
whether
the aminoaciduria
results
from
accumulation
of amino acids in the blood, or whether
blood levels are
normal
and the aminoacidnria
results
from
defective
renal
tubular
reabsorption.
Glycinui-ia,
approximately
4000 pg./nll.,
is shown in Fig. 9. Other
Fig.
kindly
1 1. Urinary
furnished
amino
by Dr.
acid
Marvin
ci mona togra nim sit owing
Armstrong,
Fels
Research
a rgin innosnee innicacidu
Institute,
Yellow
na.
Springs,
( Speci
Ohio.)
iii
en
vol.
14. No. II,
1968
METABOLIC
1059
DISORDERS
amino acids are present
in normal
concentrations.
Homocystinuria
is
shown in Fig. 10. Mild elevations
of several
other amino
acids are
present
in this specimen.
Argimmiriosuccinic
acid is seen as a double spot
to the left of the origin in Fig. 11. Concentrations
of other amino acids
are in the normal
ranges.
Citrullinuria
is shown
in Fig. 12. Since glutamine
and citrulline
migrate
to the same position
in these solvents,
identity
of citrulhine
was
confirmed
by use of PDAB
reagent
on a duplicate
two-dimensional
chromatogram.
Figure 13 shows a chromatogram
of urine from a child with a urinary
tract
infection.
acterize
this
Marked
elevation
of lysimie, cystine,
pattern
as “stone-forming”
cystinuria.
and
arginine
charBlood
levels
of
5,j
Fig. 12. Urinna ny amino
by Dr.
WI. C. MeMurray,
acid
chromatogram
Umnivensity
of
Western
showing
Ontario,
citruhlinnunia.
London,
(Specinnemi
Omit.,
kindly
Canada.)
funnishnel
BERRY FT AL.
1060
Clirdcal
Chemistry
these amitino acids are normal.
Generalized
gross arninoaciduria
in a
child with Fanconi-Lignac
syndrolne
(cystine
storage
disease)
is shown
in Fig. 14. In this instance,
only i pJ. (one-tenth
the usual amount)
of
urine was used to permit
separation
of the amino
acids. Note that
Fig.
13.
Urinary
annino acid chromatogram
with
“stone
forming”
cystinunia,
showing
excessive
ex-
cretion of hysimne, arginine, cystine,
and ormnithine.
I
cystine forms a double spot in the acidic solvent. In this disease,
excretion of tyrosine
and phiellylalanilte
are usually
normal, and blood amino
acids are normal or low.
Aminoaciduria
characteristic
of the acquired
Fanconi
syndrome
is
shown in Fig. 15. Toxic (lamnage to liver enzymes
was suspected
because
of the accumulation
of tyrosine,
phenylalanine,
and methionine,
and
the unusually
high excretion
of alanine.
While the specific toxic sub-
Vol. 14, No. II,
METABOLIC
1968
1061
DISORDERS
stance was not identified
in this instance,
similar
patterns
have been
observed
following
ingestion
of lead, mercury,
phosphorus,
and aged
tetracycline.
Blood amino acids may be elevated.
Gross aminoaciduria
characteristic
of Hartnup
disease
is shown un
Fig. 16. Tryptophan
does not separate
froni tyrosine.
Conspicuous
features are the strong
spot for ghitamine
plus citrulline,
mai’ked elevation
of leucine amid vahiime, and the relatively
low excretion
of glvcilte, lysimne,
and histidimne. Blood amuito acids are usually normal.
30
‘F0
UNNAil
Fig. 14. Urinary
amino
acid chromatogram
characteristic
of Fanconi
symidronne, associated
Chromniatogram was prepared
usimng 5-s1. urine
showing
with
(n/,0
gross
J,
generalized
giucosunia,
the amount
OIJ
aminnoaciduria,
pattern
proteiauria,
ann(l phnosphnatuni:n.
usually
applied
1062
BERRY
FT
Clinical
AL.
Chemistry
Figure
17 shows gross aminoaciduiria
in a child with Lowe’s
syndrome. Nonessential
anmino acids and basic amino acids are most conspicuous.
The marked
elevation
of prohine
is not shown
on the
ninhydrin
chromatogram.
Blood amino acids are usually
normal.
The gross
amninoacidi.iria
shown
iii Fig. 18 was associated
with
tyrosinosis.
Excretions
of pheriylalalmilme,
tvrosilte,
amid methionine
are
characteristically
elevated
to 10-20 times the normal level, while valine
amid leucine
may be only slightly
elevated.
Citrulline
and glutamine
formn a single spot and are not clearly separated
from lysine when such
large
concentrations
are present.
Blood
levels
of tyrosine,
pheiiylalanine,
and methionine
may be elevated.
________
“C’
elan
Fig.
with
15. Urinary
acquired
Fanneoni
annimno acid
syndrome.
elnromatognam
Note marked
sinowiing generalized
excretion
of alanninne.
.
OHio
amniinnoacidunia
associated
vol. 14, No. II,
1968
METABOLIC
1063
DISORDERS
Comment
The procedures
described
have evolved from use during
more than
10 years of routine screening
of urine specimens
for metabolic
disorders.
Of the many variations
which were tried, we selected
the ones most
satisfactory
from the standpoint
of reliability,
reproducibility
of results, simplicity,
and low cost. Most of the abnormalities
of amino acid
and carbohydrate
metabolism
will be recognized
in the course
of the
screening-both
those indicated
by the presence
of abnormal
substances
and those characterized
by the presence
of abnormal
amounts
of normal
constituents.
For many of the biochemical
abnormalities
thus revealed,
specific therapy
has been developed.
Although
no treatment
has been
proposed
for many metabolic
disorders,
recognition
of the etiology
can
be of great
importance
in prognosis,
management,
and
genetic
counseling.
Fig.
16. IJninary
amino
acid clnromatognamn
of Ilartmnup disease. Basic amino
amino acids. Ten microliters
(‘/
acids ainl
tine usual
sinowing
generalized
glycimnc are low compared
volunne) was applied.
amnino:neiduria
witln p;nttermn
to conneenntmationns of otlner
Fig. 17.
IJrimnary
amnimno acid chrominatogram
fronn an infant
with cataracts
and liypotonnia at birth, showing patteril
characteristic
of
dnonnne.
UIIICINAII
1$,
Lowe’s
symn.
OHIO
Fig.
nun
18.
o acid
Uninnary
chronnia to-
gnamn fronn n-mud with
tyrosiniosis.
M e tin i o aiim and algimnine are
present.
‘i’emn microliters
of
urine
was
applied.
1UHI15
Vol. 14, No. II,
1968
METABOLIC
DISORDERS
1065
References
I.
2.
3.
4.
3.
Stannbury,
J. B., Wyagnarden,
J. B.,and
Fredrickson,
D. S., The Metabolic
Bu.’i.s of
Inherited
Disease
(ed. 2). McGraw-Hill
(Blakiston),
New York, 1966.
Berry,
H. K., Suttomi, H. E., Cain, L., and Berry, J. S., “Development
of Paper
Chromnatography
for Use in the Study
of Metabolic
Pntterns.”
In Individual
Metabolic
Patterns
and Human
Disease:
An Exploratory
Study
Utilizing
Predominantly
Paper
Chromatographic
Methods.
Biochemical
Institute
Studies
IV, University
of Texas Publication
5109,
Umniv. Texas Press, Austin,
Tex., 1951.
Smith, I., Chronatographic
and Electroplnorelic
Techniques.
Chromatography
(ed. 2, Vol. I).
Interscience,
New York, 1960.
Block, B. J., Durrum,
E. L., and Zweig, G., A Manual
of Paper Clnronnatograpliy
and Paper
Electrophore.sis
(ed. 2). Acad. Press, New York, 1958.
TTmbam-ger,
B., Paper
chromatographic
method
for
quantitatiomn
of urinary
fructose
(levulose).
J. Lab. Chin. Med. 60, 521 (1962).