- Email: [email protected]
The estimation of vanilmandelic acid (VMA) in serum and urine by a radioactive 14C labelling technique
CLISICA
THE
485
CHIMICA ACTA
ESTIMATION
URINE
BY
Defxzvtnzeni (Received
OF
VANILMANDELIC
A RADIOACTIVE
ACID
l+C LABELLING
(VMA)
IN
SERUM
AND
TECHNIQUE
ofChemical Pathology, The Royal Ilzfvmavy, Shefield 6 (U.K.)
November
joth,
1967)
SUMMARY
A method is described for the estimation of vanilmandelic acid (VMA; vanillylmandelic acid; 3-methoxy-4-hydroxy mandelic acid) in urine and serum. VMA is separated from other phenolic acids by electrophoresis on cellulose thin-layer plates at a constant
potential
of 500 V. The isolated
VMA is acetylated
with radioactive
[I-l*C]acetic anhydride, and the radioactivity of the reaction product counted. Results obtained in a number of cases of phaeochromocytoma and neuroblastoma are described.
Since the identification of VMA (vanilmandelic acid, vanillylmandelic 3-methoxy-4-hydroxy mandelic acid) as the major urinary catecholamine
acid, meta-
bolitel, several methods for its estimation in urine have been described, which have been reviewed by Ruthven2 and SandleP. The group of methods involving chromatography (paper, thin-layer, and gas) and electrophoresis, have been called semiquantitative techniquess, but they offer scope for the specific analysis of VMA in urine and serum. Measurement of VMA in blood from a patient with a phaeochromocytoma by conversion to vanillin has been described”, but the amount of blood required results in the test being of little use. Gitlow5 suggested that VMA in blood might be determined by trifluoracetylation of its vanillin derivative, followed by gas chromatographic analysis of the product using electron capture detection. He quoted no results for VM4 in serum. It seemed possible that by acetylating VMA with [r-lK]acetic anhydride, a technique could be devised sensitive enough for the specific analysis of VMA in urine and serum. METHOD
Reagents ami thidayer plates Sodium carbonate: 2% w/v and 10% w/v aqueous solutions. Sodium nitrite: 10% w/v aqueous solution. Store at 4O in I ml lots. I/MA standards (Camlab Ltd.): I mg and IOO mg per IOO ml methanol. at -20’.
Store
Clin. Chinz. Acta, 19 (1968) 485-492
486
O'GORMAN
Electrophovesis buffer: 3.0 ml pyridine and 16.0 ml glacial acetic acid made up to I 1 with distilled water. pH 3.9. r_llkuEine ~,e~~~nol reagei?t: Mix 25 ml of 2y0 w/v aqueous sodium carbonate and 25 ml methanol
immediately
before use.
Store in a brown bottle6. ,&K~itroanili~ze soldow: 1.5 g p-nitroaniline
Ethyl acetate:
dissolved
in 45 ml concentrated
HCl, made up to I 1 with distilled water. I~~~~ot~~ed P-~~~t~ou~l~~~~Le reagelzt: Mix I0 ml j-nitroaniline solution, IO'?& W/V sodium nitrite solution, and IO ml 1oO.A w/v sodium carbonate immediately before use.
0.2 mI of solution,
I db Potassium carbonate: 138 g anhydrous potassium carbonate made up to I 1 with distilled water. .I-IK’]Acetic a?zhydride solutio?zs: / r-‘%],4cetic anhydride was obtained from Radio~t~er~lical Centre, Amersham. I;rCrzary l’i;11,4 acetyzat~~zg veagent:
0.1 mC (45.1 mC/mmole) of ‘Q.Zactivity
l~r-lS]acetic anhydride, diluted to IO ml with acetic anhydride. Serttpn VJIL4 acet$ating reagefzt: 0.1 mC (45.1 mC/mmole) acetic anhydride diluted to I ml with acetic anhydride.
of “C. activity
in in
CeEEztEosethidayer jdates: (MN-Cellulose 300, Macherey, Nagel & Co.) To make five zo x 20 cm plates, or three 20 x 35 cm plates, 20 e powder is ll~~nlo~~nised with IZO ml distilled water in a fast electric mixer. Plates are spread to a wet thickness of device, dried at 100~ for IO min, and stored in a desiccator over concentrated sulphuric acid.
500 mp, using a spreading
~~t~a~~~o~L.Deterlninations are carried out in quadruplicate. The creatinine content is determined, and unless very dilute, urines are shaken with Florisil to remove coloured substances. The volume of urine containing 0.5 mg creatinine, from a 24-h specimen of urine, is diluted to 2.0 ml with 0.1 X HCl, and saturated with solid sodium chloride. The solution is extracted by vigorous shaking with 14 volume of diethy ether. The extraction is repeated twice more, the ether layers combined and evaporated, and the residue taken up in IOO ,~l methanol. Elcctro$hovesis. The methanolic solutions of the urinary extracts are applied to a cellulose thin-layer plate I cm from the anode end, and electrophoresis is carried out at pH 3.9 for 3 h with a constant potential of 500 I’ (ref. 7). The VMiz travels towards the anode and separates sufficiently from other urinary phenolic acids (see Fig. I) for isolation of the VMA zone to be possible. To locate the YMA zone, one of the quadruplicate samples is sprayed with diazotized p-nitroaniline, with which t’MA forms a purple colour. The other three VMA zones are isolated by cutting out the zones with a r-inch section of a one-sided blade as shown in Fig. I. The VK4 is eluted from the cellulose by shaking with 5 ml methanol, filtering, and evaporating the methanol. The isolated VMA is taken up in 80 ,ul of urinary l_I-‘Kjacetic anhydride acetyiating reagent (0.01 mC/ml), 20 ~1 trifluoracetic acid are added as catalyst, and the mixture is heated at 80" for 30 min. The reaction is shown in Equation (I).
C&T. Chim. AGt‘Z,19 (rg6S) 485-492
487
VMA IX SERUM AKD URISE
,
.
* *
0 OCCH3 CCOOH (1)
OCHII
OCHj 3.me!hoxy-4.hydronymandelx acid (VMAl
AcetIc
anhydrIde
3.methoxy-4.acetoxy (0: acetoxy )
phenyl *
acetic acid
the acetylation of VMA with [ I-lXjacetic anhydride at So”, using trifluoracetic acid as catalyst. The diacetoxy-VMA product is taken up in approximately IOO yl of methanol and a second electrophoresis is carried out for 90 min at pH 3.9 with a constant showing
* The product of the acetylation reaction, 3-methoxy-4-acetoxy-phenpl will be referred to as diacetoxy-VJIA throughout this paper.
(wacetoxy)
Clin. Chirn. Acta, 19
acetic acid
(1~68)
485-492
488
O'GORMAN
potential of 500 V, on 20 x 35 cm plates. I ,ug and 5 pg VMA standards are acetylated with radioactive acetic anhydride and a 10-g VMA standard with non-radioactive acetic anhydride, and run on the same plate. The IO-pg standard is sprayed, after electrophoresis,
with diazotized
it acts as a marker.
$-nitroaniline
The corresponding
with which it forms a purple colour:
diacetoxy-VMA
zones are isolated
as previ-
ously described, taken up in 2 ml methanol, centrifuged, and the separated supernatant made up to IO ml with toluene. The 14C activity, expressed as counts per minute
(counts/min)
is measured
in a liquid scintillation
counter.
Calculation counts/min
test ~~~ standard
counts/min
x
,ug
Estimation of VMA in semm Extractiox. The proteins
standard
x
2 =
are precipitated
pg
VMA/mg creatinine
from 5 ml of serum by the addition
of 0.5 ml of concentrated HCl. The sample is centrifuged, is saturated with solid sodium chloride and extracted
the separated supernatant with 14 volumes of ethyl
acetate; the extraction is repeated twice more and the combined ethyl acetate layers shaken with 5 ml I M K&O,. The aqueous carbonate layer is brought to pH I by the addition of concentrated HCI and saturated with solid sodium chloride. Three extractions with I+ volumes of diethyl ether are carried out and the combined ether layers are evaporated in a 3” x I" test tube. Approximately to wash the extract from the sides down to the bottom evaporated slowly at 50~60”. Acetylation and electrophoresis The dry serum extract acetylating
reagent
is taken
(0.1 mC/ml),
2 ,d
50 ,~l of methanol are added of the tube and the methanol
up in IO ,~l of serum of trifluoracetic
L+X]acetic
anhydride
acid added, and the mixture
heated at 80” for 30 min. The acetylated products are taken up in 50 ,ul of methanol and applied as for the urinary estimation to a cellulose thin-layer plate and electrophoresis is carried out, under the previous conditions, for 24 h. A IO-,ug diacetoxyVMA standard (non-radioactive), along with IOO ng and 500 ng diacetoxy-VMA standards (radioactive), are run on the same plate. After electrophoresis, and spraying of the ro-pg standard with diazotized $-nitroaniline, the diacetoxy-VMA zones are isolated and eluted into 2 ml methanol, centrifuged, and the supernatant made up to IO ml with toluene. The 14C activity expressed as counts per minute is measured on a liquid scintillation Calculation counts/min counts/min
counter.
test standard
x
ng standard
x
20 =
ng VMA/roo
ml serum.
RESULTS
Kefwoducibility and linearity Triplicate determinations [%]diacetoxy product, using Clin.
Chim.
Acta,
19 (1968) 485-492
of standard amounts the urine acetylating
of VMA as counts/min of the reagent, 0.01 mC/ml of laC
VMArh-
SERUMAND URINE
activity
in
[r-14C]acetic
VMA (45 f
z counts/min)
489
anhydride,
yielded
a standard
curve
linear
to 15 pug VMA (1388 & 20 counts/min).
from
0.5 ,q
Triplicate
deter-
minations of standard amounts of VMA acetylated with the serum acetylating reagent, 0.1 mC/ml of 14C activity in ( r-14C]acetic anhydride, yielded a standard curve linear from 50 ng (35 & 8 counts/min) to IOOO ng (995 i_ 60 counts/min). Table I shows the urinary VMA levels (pg/mg creatinine) in relation to age as TABLE
I
URINARYvnraLEVELS(iccg/mgCreatinine)IN
RELATIONTO
AGE
ASDETERMINEDBYTHE
DESCRIBED
METHOD
VMA
Urinq
pglmg
Age gvoufi pws
we&nine
o-o.5 patit%ts
0.5-Z
2-5
20 patients
20
I
: 5 4 3
s4 5 3 0
s' 0 0 0
2
0
0
0
0
2
Cl
0
0
0
20
o+.?
5-14
OUt?Y 14
IO patients
20
0
2.1+4 4.7 + 6 6.1 * 8 8.1 --fIO IO.1 + 12 12.1 + '4 ~~-
patients
4 :
..~
patients
6 I2 2 0 0
determined by the described method. The levels obtained in the urine of normal children under z years of age concur with those described by Gitlows. Table II shows the mean of triplicate determinations of serum VMA levels obtained
in eight normotensive
patients
together
with their 24-h urinary VMA excre-
tion (mg/z4 h) on the day the sample was taken. TABLE THE
MEAN
II OFTRIPLICATE
DETERMINATIONS
TENSIVEPATIENTS,ALONGWITH SPECIMEN
WAS
THE
OFSERUM
24-h EXCRETION
VMA LE"ELS(ng/IoO VMA(mg/24h)o;v
OF
N, N, x, x, Ns N, N, N8
IN EIGHT
NORMO-
THEDAYTHEBLOOD
TAKEN ___~__
___~
Patied
ml)
Age
s2 37 25 35 24 24 59 52
Sex
&I Iv1 XI M M
F F F
Mean serum nglIo0 ml I‘+0
60 200 500 300
f
80
-c 35 _!7 87 ::: 160 I- 98 160 t 75 140 + 95 560 + 135
VMA
Urine
VMA
mgl24
h
3.2 2.9 4.4 2.9 4.8 3.0 3.0 5.0
Urine and serum levels in two cases of phaeochromocytoma, and urinary levels in three cases of neuroblastoma are shown in Table III. A comparison is made of urinary results obtained by the described method, and by colour formation with diazotized $-nitroaniline after electrophoresis’. Recovery
exfie~imeds
Because a number of steps in the analyses contain the hazard of VMA losses, recovery experiments were carried out on the following steps in the estimation: (i) Extraction of VMA from urine at pH I with diethyl ether. Clin.Chim.
Acta,
19 (1968)
485-492
(ii) Elution
of VMA from cellulose with 5 ml methanol.
(iii) Elution of diacetosy-WT.4 from cellulose with 2 ml methanol. (i) L’stractiox of PeMA ,from zwim. Determination of the mean percentage recover\- from this step in the analysis was made by s~ectro~I~{)tol~letri~ ~leterll~i~lation at 5x1) n-i;? in alkaline methanol of the purI?le colour formed by VMA with diazotized p-nitroanilinc after electrophoresis at pH 3.9 for 3 11 (ref. 7). Quantities from o.5 to IO j&g \YWI were added to volumes of urine containing 0.5 mg creatinine, extracted, and electrophoresis carried out as described. After spraying, the plates were dried and the purple \‘MX-diazotized fi-nitroaniline complex eluted into 3.0 ml of v/v metlianol~z”,
w/v Sa,CO,,
and the absorbance
recorded
against
a sprayed
cellulose
blank. The percentage mean recovery of each quantity of \‘JL% was c)oO;,. (‘ii) ~~I~~~#~L of I’JiA “f~o~}~c~~~,~~~~s~ with 5 mt .77ze~~z~7701. Deterrllinati(~n of the mean percentage recovery from this step in the analysis was carried (jut b!r comparison of \XUA levels before and after isolation from a cellulose thin-layer plate. Three thin-layer plates were set up as follows: on the first, four separate lots of V&IA, z ,~g each ; on the second, four separate lots of VMA, 5 yg each; and on the was third, four separate lots of VMA, 10 jdg eaclr, were applied. Electrophoresis carried out at pH 3.9 for 3 h at a constant potential of 500 ly. Two out of each set of four standards were stained on the plate by spraying with tliazotized j+nitroaniline, the purple complex was isolated and eluted into alkaline methanol and the absorbance recorded at 510 mkt. The remaining pair out of each set of four standards, their position on the plate having been located by the staining of the other pair, were isolated by cutting out as before, taken up in 5 ml methanol and applied to another thin-layer plate. The electrophoresis was carried out again under the previous conditions. The standards were then stained on the plate by spraying with diazotized $-nitroaniline, the purple complex was isolated and eluted into alkaline methanol, and the absorbance recorded at jro mp. The differences in absorbance between the standards undergoing two Clin.
Chum. Acta,
19 (1968)
485-492
VM,4 IN
SERUM
AKD
electrophoresis percentage (iii)
491
URISE
steps,
and those undergoing
only one, were determined
as a mean
recovery of 9501;. Elution of diacetoxy-VMA from cellulose of known amounts of LKldiacetoxy-VMA
aith 2 ml methasol. Analysis of was carried out by radioactive duplicates counting. Duplicates of identical amounts were subjected to electrophoresis at pH 3.9 with a constant potential of 500 V and the radioactive compound isolated into z ml measured. Commethanol, made up to 10 ml with toluene and the Y radioactivity parison of the two sets of results, representing loss due to isolation was determined as a mean percentage recovery of 96”,,. The total
mean
percentage
recovery
of VMA through
from the cellulose,
the whole procedure
was 84” o The spec$city
of the radioactizje
labelling
techuipe
Fig. I shows that the possibility of eluting phenolic acids other than VMX from the electrophoretic strip exists. The specificity of the technique described was studied by gas chromatography of a volatile derivative of VM4 extracted from urine and isolated from the electrophoretic cellulose strip. Methyl esterification with ethereal diazomethane solution for 5 min at room temperature, followed by acetylation with acetic anhydride as previously described, was carried out. A single peak (Pye-Argon gas chromatograph ; I o b XE 60 column on SO~IOO mesh acid-washed celite ; temperature rXo”, flash heater 220~; inlet pressure 20 p.s.i.) was obtained, with a retention of S min, identical to that obtained for standard VMA treated similarly.
time
The purity of the eluted VMA was further established by formation of another derivative, methyl (3, 4-dimethoxy) mandelate formed by treating VMA with v/v methanol-ethereal diazomethane overnight at room temperature. A single peak, under the same gas-chromatographic conditions, was obtained with a retention time of 6.5 min, identical
to that obtained
for standard
VMA treated
similarly.
DISCUSSIOX
The described method for analysis of urinary VMA is more protracted than the majority of techniques for VMA analysis described in the literature, but its sensitivity and reproducibility allow an accurate analysis of small physiological changes in the urinary excretion of VMA. Results obtained in normal and pathological cases concur with those obtained by a modification of Hermann’s technique (1964). As in Hermann’s technique, the excretion of salicyluric acid, after aspirin ingestion, interferes appreciably with the test, but since in the first step one of the quadruplicate extracts is stained with diazotized $-nitroaniline, any interfering phenolic acids can be observed to be present, before acetylation is carried out. The technique may be employed for the accurate analysis of any phenolic acid that can be satisfactorily isolated, or indeed its acetoxy derivative satisfactorily isolated by an electrophoretic or chromatographic technique. The high standard deviation obtained in the analysis of VMA in serum (Table II) is unfortunate, but may well be attributed to the relatively low concentration of lpC activity in the acetic anhydride (0.1 mC/ml). Equally, carrying out the acetylation in the relatively large 3” x I” test tubes may perhaps lead to incomplete reaction. The two abnormal serum VMA levels obtained from two preoperative cases of phaeo-
O’GORMAN
492
chromocytoma, suggest that there is hope of a greater distinction between pathological and normal serum levels than in urine. In the eight normotensives studied, some correlation between serum levels and total 24-h excretion of VMA appears to exist. ACKSOWLEDGEMENTS
I wish to thank Dr. A. Jordan for advice and encouragement during the courst of this work, Dr. P. A. Toseland for constructive criticism, Mr. C. I. Frank and Mrs. N. Hobson of Sheffield Medical Physics for assistance with the radioactive counting.
I M. I>.;\RMSTRONG, A. ibICblILLAN .
‘049.
CZzn. Chim.
,4&z,
19 (1968)
485-492
invest.,
44
THE
485
CHIMICA ACTA
ESTIMATION
URINE
BY
Defxzvtnzeni (Received
OF
VANILMANDELIC
A RADIOACTIVE
ACID
l+C LABELLING
(VMA)
IN
SERUM
AND
TECHNIQUE
ofChemical Pathology, The Royal Ilzfvmavy, Shefield 6 (U.K.)
November
joth,
1967)
SUMMARY
A method is described for the estimation of vanilmandelic acid (VMA; vanillylmandelic acid; 3-methoxy-4-hydroxy mandelic acid) in urine and serum. VMA is separated from other phenolic acids by electrophoresis on cellulose thin-layer plates at a constant
potential
of 500 V. The isolated
VMA is acetylated
with radioactive
[I-l*C]acetic anhydride, and the radioactivity of the reaction product counted. Results obtained in a number of cases of phaeochromocytoma and neuroblastoma are described.
Since the identification of VMA (vanilmandelic acid, vanillylmandelic 3-methoxy-4-hydroxy mandelic acid) as the major urinary catecholamine
acid, meta-
bolitel, several methods for its estimation in urine have been described, which have been reviewed by Ruthven2 and SandleP. The group of methods involving chromatography (paper, thin-layer, and gas) and electrophoresis, have been called semiquantitative techniquess, but they offer scope for the specific analysis of VMA in urine and serum. Measurement of VMA in blood from a patient with a phaeochromocytoma by conversion to vanillin has been described”, but the amount of blood required results in the test being of little use. Gitlow5 suggested that VMA in blood might be determined by trifluoracetylation of its vanillin derivative, followed by gas chromatographic analysis of the product using electron capture detection. He quoted no results for VM4 in serum. It seemed possible that by acetylating VMA with [r-lK]acetic anhydride, a technique could be devised sensitive enough for the specific analysis of VMA in urine and serum. METHOD
Reagents ami thidayer plates Sodium carbonate: 2% w/v and 10% w/v aqueous solutions. Sodium nitrite: 10% w/v aqueous solution. Store at 4O in I ml lots. I/MA standards (Camlab Ltd.): I mg and IOO mg per IOO ml methanol. at -20’.
Store
Clin. Chinz. Acta, 19 (1968) 485-492
486
O'GORMAN
Electrophovesis buffer: 3.0 ml pyridine and 16.0 ml glacial acetic acid made up to I 1 with distilled water. pH 3.9. r_llkuEine ~,e~~~nol reagei?t: Mix 25 ml of 2y0 w/v aqueous sodium carbonate and 25 ml methanol
immediately
before use.
Store in a brown bottle6. ,&K~itroanili~ze soldow: 1.5 g p-nitroaniline
Ethyl acetate:
dissolved
in 45 ml concentrated
HCl, made up to I 1 with distilled water. I~~~~ot~~ed P-~~~t~ou~l~~~~Le reagelzt: Mix I0 ml j-nitroaniline solution, IO'?& W/V sodium nitrite solution, and IO ml 1oO.A w/v sodium carbonate immediately before use.
0.2 mI of solution,
I db Potassium carbonate: 138 g anhydrous potassium carbonate made up to I 1 with distilled water. .I-IK’]Acetic a?zhydride solutio?zs: / r-‘%],4cetic anhydride was obtained from Radio~t~er~lical Centre, Amersham. I;rCrzary l’i;11,4 acetyzat~~zg veagent:
0.1 mC (45.1 mC/mmole) of ‘Q.Zactivity
l~r-lS]acetic anhydride, diluted to IO ml with acetic anhydride. Serttpn VJIL4 acet$ating reagefzt: 0.1 mC (45.1 mC/mmole) acetic anhydride diluted to I ml with acetic anhydride.
of “C. activity
in in
CeEEztEosethidayer jdates: (MN-Cellulose 300, Macherey, Nagel & Co.) To make five zo x 20 cm plates, or three 20 x 35 cm plates, 20 e powder is ll~~nlo~~nised with IZO ml distilled water in a fast electric mixer. Plates are spread to a wet thickness of device, dried at 100~ for IO min, and stored in a desiccator over concentrated sulphuric acid.
500 mp, using a spreading
~~t~a~~~o~L.Deterlninations are carried out in quadruplicate. The creatinine content is determined, and unless very dilute, urines are shaken with Florisil to remove coloured substances. The volume of urine containing 0.5 mg creatinine, from a 24-h specimen of urine, is diluted to 2.0 ml with 0.1 X HCl, and saturated with solid sodium chloride. The solution is extracted by vigorous shaking with 14 volume of diethy ether. The extraction is repeated twice more, the ether layers combined and evaporated, and the residue taken up in IOO ,~l methanol. Elcctro$hovesis. The methanolic solutions of the urinary extracts are applied to a cellulose thin-layer plate I cm from the anode end, and electrophoresis is carried out at pH 3.9 for 3 h with a constant potential of 500 I’ (ref. 7). The VMiz travels towards the anode and separates sufficiently from other urinary phenolic acids (see Fig. I) for isolation of the VMA zone to be possible. To locate the YMA zone, one of the quadruplicate samples is sprayed with diazotized p-nitroaniline, with which t’MA forms a purple colour. The other three VMA zones are isolated by cutting out the zones with a r-inch section of a one-sided blade as shown in Fig. I. The VK4 is eluted from the cellulose by shaking with 5 ml methanol, filtering, and evaporating the methanol. The isolated VMA is taken up in 80 ,ul of urinary l_I-‘Kjacetic anhydride acetyiating reagent (0.01 mC/ml), 20 ~1 trifluoracetic acid are added as catalyst, and the mixture is heated at 80" for 30 min. The reaction is shown in Equation (I).
C&T. Chim. AGt‘Z,19 (rg6S) 485-492
487
VMA IX SERUM AKD URISE
,
.
* *
0 OCCH3 CCOOH (1)
OCHII
OCHj 3.me!hoxy-4.hydronymandelx acid (VMAl
AcetIc
anhydrIde
3.methoxy-4.acetoxy (0: acetoxy )
phenyl *
acetic acid
the acetylation of VMA with [ I-lXjacetic anhydride at So”, using trifluoracetic acid as catalyst. The diacetoxy-VMA product is taken up in approximately IOO yl of methanol and a second electrophoresis is carried out for 90 min at pH 3.9 with a constant showing
* The product of the acetylation reaction, 3-methoxy-4-acetoxy-phenpl will be referred to as diacetoxy-VJIA throughout this paper.
(wacetoxy)
Clin. Chirn. Acta, 19
acetic acid
(1~68)
485-492
488
O'GORMAN
potential of 500 V, on 20 x 35 cm plates. I ,ug and 5 pg VMA standards are acetylated with radioactive acetic anhydride and a 10-g VMA standard with non-radioactive acetic anhydride, and run on the same plate. The IO-pg standard is sprayed, after electrophoresis,
with diazotized
it acts as a marker.
$-nitroaniline
The corresponding
with which it forms a purple colour:
diacetoxy-VMA
zones are isolated
as previ-
ously described, taken up in 2 ml methanol, centrifuged, and the separated supernatant made up to IO ml with toluene. The 14C activity, expressed as counts per minute
(counts/min)
is measured
in a liquid scintillation
counter.
Calculation counts/min
test ~~~ standard
counts/min
x
,ug
Estimation of VMA in semm Extractiox. The proteins
standard
x
2 =
are precipitated
pg
VMA/mg creatinine
from 5 ml of serum by the addition
of 0.5 ml of concentrated HCl. The sample is centrifuged, is saturated with solid sodium chloride and extracted
the separated supernatant with 14 volumes of ethyl
acetate; the extraction is repeated twice more and the combined ethyl acetate layers shaken with 5 ml I M K&O,. The aqueous carbonate layer is brought to pH I by the addition of concentrated HCI and saturated with solid sodium chloride. Three extractions with I+ volumes of diethyl ether are carried out and the combined ether layers are evaporated in a 3” x I" test tube. Approximately to wash the extract from the sides down to the bottom evaporated slowly at 50~60”. Acetylation and electrophoresis The dry serum extract acetylating
reagent
is taken
(0.1 mC/ml),
2 ,d
50 ,~l of methanol are added of the tube and the methanol
up in IO ,~l of serum of trifluoracetic
L+X]acetic
anhydride
acid added, and the mixture
heated at 80” for 30 min. The acetylated products are taken up in 50 ,ul of methanol and applied as for the urinary estimation to a cellulose thin-layer plate and electrophoresis is carried out, under the previous conditions, for 24 h. A IO-,ug diacetoxyVMA standard (non-radioactive), along with IOO ng and 500 ng diacetoxy-VMA standards (radioactive), are run on the same plate. After electrophoresis, and spraying of the ro-pg standard with diazotized $-nitroaniline, the diacetoxy-VMA zones are isolated and eluted into 2 ml methanol, centrifuged, and the supernatant made up to IO ml with toluene. The 14C activity expressed as counts per minute is measured on a liquid scintillation Calculation counts/min counts/min
counter.
test standard
x
ng standard
x
20 =
ng VMA/roo
ml serum.
RESULTS
Kefwoducibility and linearity Triplicate determinations [%]diacetoxy product, using Clin.
Chim.
Acta,
19 (1968) 485-492
of standard amounts the urine acetylating
of VMA as counts/min of the reagent, 0.01 mC/ml of laC
VMArh-
SERUMAND URINE
activity
in
[r-14C]acetic
VMA (45 f
z counts/min)
489
anhydride,
yielded
a standard
curve
linear
to 15 pug VMA (1388 & 20 counts/min).
from
0.5 ,q
Triplicate
deter-
minations of standard amounts of VMA acetylated with the serum acetylating reagent, 0.1 mC/ml of 14C activity in ( r-14C]acetic anhydride, yielded a standard curve linear from 50 ng (35 & 8 counts/min) to IOOO ng (995 i_ 60 counts/min). Table I shows the urinary VMA levels (pg/mg creatinine) in relation to age as TABLE
I
URINARYvnraLEVELS(iccg/mgCreatinine)IN
RELATIONTO
AGE
ASDETERMINEDBYTHE
DESCRIBED
METHOD
VMA
Urinq
pglmg
Age gvoufi pws
we&nine
o-o.5 patit%ts
0.5-Z
2-5
20 patients
20
I
: 5 4 3
s4 5 3 0
s' 0 0 0
2
0
0
0
0
2
Cl
0
0
0
20
o+.?
5-14
OUt?Y 14
IO patients
20
0
2.1+4 4.7 + 6 6.1 * 8 8.1 --fIO IO.1 + 12 12.1 + '4 ~~-
patients
4 :
..~
patients
6 I2 2 0 0
determined by the described method. The levels obtained in the urine of normal children under z years of age concur with those described by Gitlows. Table II shows the mean of triplicate determinations of serum VMA levels obtained
in eight normotensive
patients
together
with their 24-h urinary VMA excre-
tion (mg/z4 h) on the day the sample was taken. TABLE THE
MEAN
II OFTRIPLICATE
DETERMINATIONS
TENSIVEPATIENTS,ALONGWITH SPECIMEN
WAS
THE
OFSERUM
24-h EXCRETION
VMA LE"ELS(ng/IoO VMA(mg/24h)o;v
OF
N, N, x, x, Ns N, N, N8
IN EIGHT
NORMO-
THEDAYTHEBLOOD
TAKEN ___~__
___~
Patied
ml)
Age
s2 37 25 35 24 24 59 52
Sex
&I Iv1 XI M M
F F F
Mean serum nglIo0 ml I‘+0
60 200 500 300
f
80
-c 35 _!7 87 ::: 160 I- 98 160 t 75 140 + 95 560 + 135
VMA
Urine
VMA
mgl24
h
3.2 2.9 4.4 2.9 4.8 3.0 3.0 5.0
Urine and serum levels in two cases of phaeochromocytoma, and urinary levels in three cases of neuroblastoma are shown in Table III. A comparison is made of urinary results obtained by the described method, and by colour formation with diazotized $-nitroaniline after electrophoresis’. Recovery
exfie~imeds
Because a number of steps in the analyses contain the hazard of VMA losses, recovery experiments were carried out on the following steps in the estimation: (i) Extraction of VMA from urine at pH I with diethyl ether. Clin.Chim.
Acta,
19 (1968)
485-492
(ii) Elution
of VMA from cellulose with 5 ml methanol.
(iii) Elution of diacetosy-WT.4 from cellulose with 2 ml methanol. (i) L’stractiox of PeMA ,from zwim. Determination of the mean percentage recover\- from this step in the analysis was made by s~ectro~I~{)tol~letri~ ~leterll~i~lation at 5x1) n-i;? in alkaline methanol of the purI?le colour formed by VMA with diazotized p-nitroanilinc after electrophoresis at pH 3.9 for 3 11 (ref. 7). Quantities from o.5 to IO j&g \YWI were added to volumes of urine containing 0.5 mg creatinine, extracted, and electrophoresis carried out as described. After spraying, the plates were dried and the purple \‘MX-diazotized fi-nitroaniline complex eluted into 3.0 ml of v/v metlianol~z”,
w/v Sa,CO,,
and the absorbance
recorded
against
a sprayed
cellulose
blank. The percentage mean recovery of each quantity of \‘JL% was c)oO;,. (‘ii) ~~I~~~#~L of I’JiA “f~o~}~c~~~,~~~~s~ with 5 mt .77ze~~z~7701. Deterrllinati(~n of the mean percentage recovery from this step in the analysis was carried (jut b!r comparison of \XUA levels before and after isolation from a cellulose thin-layer plate. Three thin-layer plates were set up as follows: on the first, four separate lots of V&IA, z ,~g each ; on the second, four separate lots of VMA, 5 yg each; and on the was third, four separate lots of VMA, 10 jdg eaclr, were applied. Electrophoresis carried out at pH 3.9 for 3 h at a constant potential of 500 ly. Two out of each set of four standards were stained on the plate by spraying with tliazotized j+nitroaniline, the purple complex was isolated and eluted into alkaline methanol and the absorbance recorded at 510 mkt. The remaining pair out of each set of four standards, their position on the plate having been located by the staining of the other pair, were isolated by cutting out as before, taken up in 5 ml methanol and applied to another thin-layer plate. The electrophoresis was carried out again under the previous conditions. The standards were then stained on the plate by spraying with diazotized $-nitroaniline, the purple complex was isolated and eluted into alkaline methanol, and the absorbance recorded at jro mp. The differences in absorbance between the standards undergoing two Clin.
Chum. Acta,
19 (1968)
485-492
VM,4 IN
SERUM
AKD
electrophoresis percentage (iii)
491
URISE
steps,
and those undergoing
only one, were determined
as a mean
recovery of 9501;. Elution of diacetoxy-VMA from cellulose of known amounts of LKldiacetoxy-VMA
aith 2 ml methasol. Analysis of was carried out by radioactive duplicates counting. Duplicates of identical amounts were subjected to electrophoresis at pH 3.9 with a constant potential of 500 V and the radioactive compound isolated into z ml measured. Commethanol, made up to 10 ml with toluene and the Y radioactivity parison of the two sets of results, representing loss due to isolation was determined as a mean percentage recovery of 96”,,. The total
mean
percentage
recovery
of VMA through
from the cellulose,
the whole procedure
was 84” o The spec$city
of the radioactizje
labelling
techuipe
Fig. I shows that the possibility of eluting phenolic acids other than VMX from the electrophoretic strip exists. The specificity of the technique described was studied by gas chromatography of a volatile derivative of VM4 extracted from urine and isolated from the electrophoretic cellulose strip. Methyl esterification with ethereal diazomethane solution for 5 min at room temperature, followed by acetylation with acetic anhydride as previously described, was carried out. A single peak (Pye-Argon gas chromatograph ; I o b XE 60 column on SO~IOO mesh acid-washed celite ; temperature rXo”, flash heater 220~; inlet pressure 20 p.s.i.) was obtained, with a retention of S min, identical to that obtained for standard VMA treated similarly.
time
The purity of the eluted VMA was further established by formation of another derivative, methyl (3, 4-dimethoxy) mandelate formed by treating VMA with v/v methanol-ethereal diazomethane overnight at room temperature. A single peak, under the same gas-chromatographic conditions, was obtained with a retention time of 6.5 min, identical
to that obtained
for standard
VMA treated
similarly.
DISCUSSIOX
The described method for analysis of urinary VMA is more protracted than the majority of techniques for VMA analysis described in the literature, but its sensitivity and reproducibility allow an accurate analysis of small physiological changes in the urinary excretion of VMA. Results obtained in normal and pathological cases concur with those obtained by a modification of Hermann’s technique (1964). As in Hermann’s technique, the excretion of salicyluric acid, after aspirin ingestion, interferes appreciably with the test, but since in the first step one of the quadruplicate extracts is stained with diazotized $-nitroaniline, any interfering phenolic acids can be observed to be present, before acetylation is carried out. The technique may be employed for the accurate analysis of any phenolic acid that can be satisfactorily isolated, or indeed its acetoxy derivative satisfactorily isolated by an electrophoretic or chromatographic technique. The high standard deviation obtained in the analysis of VMA in serum (Table II) is unfortunate, but may well be attributed to the relatively low concentration of lpC activity in the acetic anhydride (0.1 mC/ml). Equally, carrying out the acetylation in the relatively large 3” x I” test tubes may perhaps lead to incomplete reaction. The two abnormal serum VMA levels obtained from two preoperative cases of phaeo-
O’GORMAN
492
chromocytoma, suggest that there is hope of a greater distinction between pathological and normal serum levels than in urine. In the eight normotensives studied, some correlation between serum levels and total 24-h excretion of VMA appears to exist. ACKSOWLEDGEMENTS
I wish to thank Dr. A. Jordan for advice and encouragement during the courst of this work, Dr. P. A. Toseland for constructive criticism, Mr. C. I. Frank and Mrs. N. Hobson of Sheffield Medical Physics for assistance with the radioactive counting.
I M. I>.;\RMSTRONG, A. ibICblILLAN .
‘049.
CZzn. Chim.
,4&z,
19 (1968)
485-492
invest.,
44