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Vanilmandelic acid (VMA), free and conjugated 3-methoxy-4-hydroxyphenylglycol (MHPG) in human ventricular fluid
451
Clinica Chimica Acta, 62 (1975) 451-455 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
SHORT COMMUNICATION
CCA 7235
VANILMANDELIC ACID (VMA), FliEE AND CONJUGATED 3-METHOXY-4-HYDROXYPHENYLGLYCOL (MHPG) IN HUMAN VENTRICULAR FLUID
FAROUK KAROUM, JOHN CHRISTIAN RICHARD JED WYATT
GILLIN,
Laboratory Washington, Washington,
Saint Elizabeths Hospital, SMR, IRP, NIMH, of Neurosurgery, Georgetown University,
(Received
of Clinical Psychopharmacology, D.C. 20032 and aDepartment D.C. (U.S.A.)
February
DAVID McCULLOUGHa
and
28, 1975)
Free 3-methoxy-4-hydroxyphenylglycol (MHPG) and its sulfate conjugate (MHPG-S04) are generally considered to be the major metabolites of norepinephrine (NE) in the central nervous system (CNS) [l--4]. Although vanilmandelic acid (VMA) is the main urinary metabolite of NE [ 51, little is known about its presence or possible importance in the CNS [6]. The absence of VMA reported by Sugden in the brain after intraventricular [ ’ 4 C] NE administration and Eccleston [7] coupled with the observations of Mannarino et al., 1963 and of Watson et al. [S-9] on VMA production in the brain and cerebrospinal fluid led to the assumption that VMA is a minor metabolite of CNS NE and, as such, is of little importance. This assumption, however, is at variance with that put forward by Chase et al. (1971) [lo] who suggested that VMA might be an important intraneuronal metabolite of NE in the CNS. In their studies on the excretion of VMA and MHPG in dogs, it was observed that the amount of radioactive VMA excreted is more than six times greater than that of MHPG following intraventricular administration of [ ’ 4 C] dopamine. The reverse was found when [ 3H] NE was administered. In this communication, we present data on ventricular fluid concentrations of VMA, free MHPG and MHPG-SO4 in man, which provide some quantitative information on the role of VMA in human CNS catecholamine metabolism. Ventricular fluid was obtained during neurosurgical procedures in nine patients (5 males, 4 females, age range from infancy to 75 years) who had been treated with right ventricular-right atria1 shunts for hydrocephalus or for presenile dementia associated with normal pressure hydrocephalus [ 1 l] . VMA and MHPG were assayed by mass fragmentography (MF) employing the methyl
ester-pentafluoropropionate (ME/PFP) derivative for VMA and the pt~nl;~fluoropropionate (PFP) derivative for MHPG 1121. VMA was extracted twice into ethyl acetate (10 ml) from 0.5 ml ventricular fluid acidified with 1 ml 1 N HCl. After centrifugation, aliquots from the extracts (8 and 10 ml, respectively) were combined and evaporated to dryness. The residue was dissolved in 0.3 ml of ethyl acetate, 0.2 ml was then transferred to a 1 ml Microflex tube (Kontes Glass Company, Evanston, Illinois) and dried under N *. One hundred ~1 of a 20% HCl solution in methanol was added and the mixture left to stand at room temperature for 5 minutes. The HCl/ methanol solution was prepared by mixing 1 ml acetyl chloride dropwise with 4 ml lipopure methanol (Applied Science Laboratory, Inc., Pennsylvania). The methyl ester, thus prepared, was dried under NT then acylated to the PFP derivative by heating at 70°C with 50 ~1 of a 10% solution of pentafluoropriopionyl imidazole (Pierce Chemical Company, Rockford, Ill. I in ethyl acetate for 10 minutes. The extraction and derivatization procedures described, offer optimum quantitative conditions for the assay of VMA where sub-nanogram quantities in aqueous solutions were easily detected and measured. Free MHPG was measured by extracting 0.5 ml ventricular fluid in 1 ml 1 M acetate buffer pH 6.2 with ethyl acetate and then treated as described for VMA, except that the dried extract in the microflex tube was not methylated and the PFP derivative was prepared with pentafluoropropionic anhydride (PFPA) as previously described [la]. The PFP derivative was dissolved in 20 yl ethyl acetate. The use of pentafluoropropionic anhydride for the alcoholic metaboiites consistently gave better yield of products than did pentafluoropionyl imidazole. The opposite was true for the acidic metabolites. To measure conjugated MHPG, 0.5 ml of ventricular fluid was acidified with 1 ml of 0.02 N HCl and extracted twice with diethyl ether. The ether layer was discarded after each extraction. After discarding the ether following the second extraction, the aqueous solution was heated to 70°C for two minutes and the remaining ether was blown off under N *. Following this purification procedure, 1 ml of 1 M acetate buffer pH 6.2 was added and the mixture hydrolyzed overnight with 0.2 ml of a high molecular weight fraction of glusalase [12] at 40°C. The glusalase hydrolyzes both the sulfate and glucuronide conjugates. A blank sample was also included at this stage containing 1 ml acetate buffer and 0.2 ml glusalase. After hydrolysis the conjugated MHPG was extracted and derivatized as described for the free MHPG. The glusalase employed was found to contain about 2 ng MHPG/ml. The blank was thus employed to correct for this source of contamination. All analyses were carried out in duplicate, with one of the duplicates containing a known amount of internal standard (5-10 ng). For the assay of conjugated MHPG, MHPG-SO4 standard was used instead of the free MHPG. Deuterated VMA and MHPG (VMA-d 3 and MHPG-d 3, respectively) were added to all samples before they were extracted. VMA-d 3 was prepared by exchanging VMA with ‘HCl and MHPG-d 3 prepared by reducing VMA-d 3 with boranemethyl sulfide (BMS). These procedures will be published in detail elsewhere
[131. Fig. 1 shows the multiple ion detection (MID) of ventricular VMA as well as that of authentic VMA. MID is a technique whereby the mass spectrometer
453
w
4 0
4.4
WIN
Fig. 1. Multiple ion detection (MID) of the methyl ester-pentafluoropropionate derivative of VMA in ventricular fluid (left) and of the authentic compound (right). Solid line represents the trace produced by focusing on m/e 41’7 and the broken line that produced by focusing on mfe 445. A Finnigan Model 3000D Quadnzpole gas chromatograph mass spectrometer was used employing an 8 ft X l/8 inch i.d. 3% SE 54 steel column. The temperature was maintained isothermally at 195°C.
is focused simultaneously on two or more fragments of the metabolite in question in order to determine the ratios of the intensities of the fragments against one another, and to compare these ratios with those of the authentic compound. The closeness in the ratios between the biological metabolite and the authentic standard offer two important pieces of info~ation. First, they confirm the identity and purity of the peak corresponding to the biological metabolite and second they establish the specificity of the fragments if selected for MF. Fig. 2 illustrates typical mass fragmentograms of VMA from 0.5 ml ventricular fluid. Identities of endogenous VMA and MHPG were confirmed by MID, focusing on m/e 417 and 445 for VMA (see Fig. l), and on 311 and 458 for MHPG. For quantification by MF, fragments with m/e 445 and 458 were employed for VMA and MHPG, respectively. The mean (+S.E.M.) concentration of VMA was 3.08 * 0.60 ng/ml. The levels of free and conjugated MHPG were 11.7 it 1 and 4.67 + 1.3 ng/ml, respectively. These latter values were similar to those reported by Chase et al.
VENTRICULAR VMA
*.d
. . .
___--
f LUID
*
1,,.,,., ,
4.6
Ml1
Fig. 2. Typical mass fragmentogram of the methyl ester-pentafluoroproprionate derivative of VMA in ventricular fluid. Solid line was produced by focusing on m/c 445 (fragment corresponding to VMA) and the broken line was produced by focusing on m/e 448, (VMA-d3). Mass spectrometer and GC conditions are as mentioned in Fig. 1. The fragmentogram on the right is that of a duplicate containing 5 ng VMA internal standard (see text).
(1973) [14] who observed ventricular fluid values of free and conjugated MHPG of 9.4 -t 3.7 and 2.7 f 1.6 ng/ml, respectively, in six patients with various CNS disorders. Thus, the present data suggest that the concentration of VMA is about 16% of total MHPG. Recently we had the oppo~unity to analyze lumbar CSF form VMA from 5 patients. The concentration observed was 0.46 + 0.18 ng/ml. Therefore, there seems to be a concentration gradient of VMA from the ventricles to the lumbar space. The physiological significance of VMA in ventricular fluid or of the relative concentrations of VMA, free MHPG, and of conjugated MHPG in CSF is not known. VMA is presumably a metabolite from NE, although some may be derived from epinephrine. Furthermore, there may be regional variations in the metabolism of NE favoring the production of VMA in some areas and of MHPG in others. Regional differences in aldehyde dehydrogenase and oxidase ]15] support such a hypothesis. Little is currently known about the relative rates of
455
release into and removal from CSF of these metabolites. Work is currently in progress in the experimental animal to study these issues. While MHPG is largely unconjugated in ventricular fluid, it is mostly excreted as MHPG-SO4 and MHPG-glucuronide in urine [12]. Whether the free MHPG coming from CNS is preferentially sulfated or glucuronidated is yet to be determined. These results do suggest, however, that VMA may be an important catecholamine metabolite in man. References 1
E. Mannarino,
N. Kirshner,
2
C.O.
Rutledge
and D.L.
-3
J.W.
Maas
4
S.M.
Schanberg,
and
5
M.D.
Armstrong,
6
E.K.
Gordon,
7
R.F.
Sugden
8
S.
Wilk
9
E. Watson,
Landis,
and
A.
J. Pharmacol.
K.
E.
Watson New
Black
S. Wilk G.R.
Chase,
Breese,
11
A.E.
Slaby
12
F. Karoum,
H. LeFevre,
13
F. Karoum.
J.C.
14
T.N.
Chase,
E.K.
15
G.V.
Erwin
New
York,
R.J.
E.K.
Wyatt
Gillin
in E.
E.
and
Gordon Usdin p. 161
Anal.
Gordon Bigelow
R.J.
and and
Wyatt,
L.K.Y. S.H.
163
(1968)
Biochem. 18
(1971)
(1963)
(1967)
I.J. Kopin, Biochim.
S.H.
10
157
373
493 147
Biochem.
Pharmacol.,
Biophys.
Acta.
Med.,
(1974)
11
25
17
(1957)
(1968)
247
422
32
2461
Snyder
(eds).
Frontiers
in
Catecholamine
Research.
p. 1067 Biochem., and
in Dementia L.B.
Ther..
Kopin,
and
Ther.,
and
Shaw,
I.J.
Usdin
1973.
and J. Roboz,
T.N.
EXP.
Breese
K.N.F.
and
Jr. J. Neurochem. EXP.
J. Neurochem..
in
York,
10
1973.
and
D. Eccleston.
Press,
and
.G.R.
McMiUan
J. Oliver,
Nashold,
J. Pharmacol.
J.J. Schidkraut,
and
and
Pergamon
B.S.
J. Jonason,
59
I.J. Kopin,
(1974)
in the Presenium, and
E.Costa,
Snyder
Charles
Clin.
J. Neurochem., Ng,
Chin
18
(1971)
C. Thomas,
Acta,
43
135
Springfield,
(1973)
Ill.,
1974
127
submitted
J. Neurochem.. (eds),
441
J. Neurochem,
Frontiers
21
(1973)
581
in Catecholamine
Research.
Pergamon
Press,
Clinica Chimica Acta, 62 (1975) 451-455 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
SHORT COMMUNICATION
CCA 7235
VANILMANDELIC ACID (VMA), FliEE AND CONJUGATED 3-METHOXY-4-HYDROXYPHENYLGLYCOL (MHPG) IN HUMAN VENTRICULAR FLUID
FAROUK KAROUM, JOHN CHRISTIAN RICHARD JED WYATT
GILLIN,
Laboratory Washington, Washington,
Saint Elizabeths Hospital, SMR, IRP, NIMH, of Neurosurgery, Georgetown University,
(Received
of Clinical Psychopharmacology, D.C. 20032 and aDepartment D.C. (U.S.A.)
February
DAVID McCULLOUGHa
and
28, 1975)
Free 3-methoxy-4-hydroxyphenylglycol (MHPG) and its sulfate conjugate (MHPG-S04) are generally considered to be the major metabolites of norepinephrine (NE) in the central nervous system (CNS) [l--4]. Although vanilmandelic acid (VMA) is the main urinary metabolite of NE [ 51, little is known about its presence or possible importance in the CNS [6]. The absence of VMA reported by Sugden in the brain after intraventricular [ ’ 4 C] NE administration and Eccleston [7] coupled with the observations of Mannarino et al., 1963 and of Watson et al. [S-9] on VMA production in the brain and cerebrospinal fluid led to the assumption that VMA is a minor metabolite of CNS NE and, as such, is of little importance. This assumption, however, is at variance with that put forward by Chase et al. (1971) [lo] who suggested that VMA might be an important intraneuronal metabolite of NE in the CNS. In their studies on the excretion of VMA and MHPG in dogs, it was observed that the amount of radioactive VMA excreted is more than six times greater than that of MHPG following intraventricular administration of [ ’ 4 C] dopamine. The reverse was found when [ 3H] NE was administered. In this communication, we present data on ventricular fluid concentrations of VMA, free MHPG and MHPG-SO4 in man, which provide some quantitative information on the role of VMA in human CNS catecholamine metabolism. Ventricular fluid was obtained during neurosurgical procedures in nine patients (5 males, 4 females, age range from infancy to 75 years) who had been treated with right ventricular-right atria1 shunts for hydrocephalus or for presenile dementia associated with normal pressure hydrocephalus [ 1 l] . VMA and MHPG were assayed by mass fragmentography (MF) employing the methyl
ester-pentafluoropropionate (ME/PFP) derivative for VMA and the pt~nl;~fluoropropionate (PFP) derivative for MHPG 1121. VMA was extracted twice into ethyl acetate (10 ml) from 0.5 ml ventricular fluid acidified with 1 ml 1 N HCl. After centrifugation, aliquots from the extracts (8 and 10 ml, respectively) were combined and evaporated to dryness. The residue was dissolved in 0.3 ml of ethyl acetate, 0.2 ml was then transferred to a 1 ml Microflex tube (Kontes Glass Company, Evanston, Illinois) and dried under N *. One hundred ~1 of a 20% HCl solution in methanol was added and the mixture left to stand at room temperature for 5 minutes. The HCl/ methanol solution was prepared by mixing 1 ml acetyl chloride dropwise with 4 ml lipopure methanol (Applied Science Laboratory, Inc., Pennsylvania). The methyl ester, thus prepared, was dried under NT then acylated to the PFP derivative by heating at 70°C with 50 ~1 of a 10% solution of pentafluoropriopionyl imidazole (Pierce Chemical Company, Rockford, Ill. I in ethyl acetate for 10 minutes. The extraction and derivatization procedures described, offer optimum quantitative conditions for the assay of VMA where sub-nanogram quantities in aqueous solutions were easily detected and measured. Free MHPG was measured by extracting 0.5 ml ventricular fluid in 1 ml 1 M acetate buffer pH 6.2 with ethyl acetate and then treated as described for VMA, except that the dried extract in the microflex tube was not methylated and the PFP derivative was prepared with pentafluoropropionic anhydride (PFPA) as previously described [la]. The PFP derivative was dissolved in 20 yl ethyl acetate. The use of pentafluoropropionic anhydride for the alcoholic metaboiites consistently gave better yield of products than did pentafluoropionyl imidazole. The opposite was true for the acidic metabolites. To measure conjugated MHPG, 0.5 ml of ventricular fluid was acidified with 1 ml of 0.02 N HCl and extracted twice with diethyl ether. The ether layer was discarded after each extraction. After discarding the ether following the second extraction, the aqueous solution was heated to 70°C for two minutes and the remaining ether was blown off under N *. Following this purification procedure, 1 ml of 1 M acetate buffer pH 6.2 was added and the mixture hydrolyzed overnight with 0.2 ml of a high molecular weight fraction of glusalase [12] at 40°C. The glusalase hydrolyzes both the sulfate and glucuronide conjugates. A blank sample was also included at this stage containing 1 ml acetate buffer and 0.2 ml glusalase. After hydrolysis the conjugated MHPG was extracted and derivatized as described for the free MHPG. The glusalase employed was found to contain about 2 ng MHPG/ml. The blank was thus employed to correct for this source of contamination. All analyses were carried out in duplicate, with one of the duplicates containing a known amount of internal standard (5-10 ng). For the assay of conjugated MHPG, MHPG-SO4 standard was used instead of the free MHPG. Deuterated VMA and MHPG (VMA-d 3 and MHPG-d 3, respectively) were added to all samples before they were extracted. VMA-d 3 was prepared by exchanging VMA with ‘HCl and MHPG-d 3 prepared by reducing VMA-d 3 with boranemethyl sulfide (BMS). These procedures will be published in detail elsewhere
[131. Fig. 1 shows the multiple ion detection (MID) of ventricular VMA as well as that of authentic VMA. MID is a technique whereby the mass spectrometer
453
w
4 0
4.4
WIN
Fig. 1. Multiple ion detection (MID) of the methyl ester-pentafluoropropionate derivative of VMA in ventricular fluid (left) and of the authentic compound (right). Solid line represents the trace produced by focusing on m/e 41’7 and the broken line that produced by focusing on mfe 445. A Finnigan Model 3000D Quadnzpole gas chromatograph mass spectrometer was used employing an 8 ft X l/8 inch i.d. 3% SE 54 steel column. The temperature was maintained isothermally at 195°C.
is focused simultaneously on two or more fragments of the metabolite in question in order to determine the ratios of the intensities of the fragments against one another, and to compare these ratios with those of the authentic compound. The closeness in the ratios between the biological metabolite and the authentic standard offer two important pieces of info~ation. First, they confirm the identity and purity of the peak corresponding to the biological metabolite and second they establish the specificity of the fragments if selected for MF. Fig. 2 illustrates typical mass fragmentograms of VMA from 0.5 ml ventricular fluid. Identities of endogenous VMA and MHPG were confirmed by MID, focusing on m/e 417 and 445 for VMA (see Fig. l), and on 311 and 458 for MHPG. For quantification by MF, fragments with m/e 445 and 458 were employed for VMA and MHPG, respectively. The mean (+S.E.M.) concentration of VMA was 3.08 * 0.60 ng/ml. The levels of free and conjugated MHPG were 11.7 it 1 and 4.67 + 1.3 ng/ml, respectively. These latter values were similar to those reported by Chase et al.
VENTRICULAR VMA
*.d
. . .
___--
f LUID
*
1,,.,,., ,
4.6
Ml1
Fig. 2. Typical mass fragmentogram of the methyl ester-pentafluoroproprionate derivative of VMA in ventricular fluid. Solid line was produced by focusing on m/c 445 (fragment corresponding to VMA) and the broken line was produced by focusing on m/e 448, (VMA-d3). Mass spectrometer and GC conditions are as mentioned in Fig. 1. The fragmentogram on the right is that of a duplicate containing 5 ng VMA internal standard (see text).
(1973) [14] who observed ventricular fluid values of free and conjugated MHPG of 9.4 -t 3.7 and 2.7 f 1.6 ng/ml, respectively, in six patients with various CNS disorders. Thus, the present data suggest that the concentration of VMA is about 16% of total MHPG. Recently we had the oppo~unity to analyze lumbar CSF form VMA from 5 patients. The concentration observed was 0.46 + 0.18 ng/ml. Therefore, there seems to be a concentration gradient of VMA from the ventricles to the lumbar space. The physiological significance of VMA in ventricular fluid or of the relative concentrations of VMA, free MHPG, and of conjugated MHPG in CSF is not known. VMA is presumably a metabolite from NE, although some may be derived from epinephrine. Furthermore, there may be regional variations in the metabolism of NE favoring the production of VMA in some areas and of MHPG in others. Regional differences in aldehyde dehydrogenase and oxidase ]15] support such a hypothesis. Little is currently known about the relative rates of
455
release into and removal from CSF of these metabolites. Work is currently in progress in the experimental animal to study these issues. While MHPG is largely unconjugated in ventricular fluid, it is mostly excreted as MHPG-SO4 and MHPG-glucuronide in urine [12]. Whether the free MHPG coming from CNS is preferentially sulfated or glucuronidated is yet to be determined. These results do suggest, however, that VMA may be an important catecholamine metabolite in man. References 1
E. Mannarino,
N. Kirshner,
2
C.O.
Rutledge
and D.L.
-3
J.W.
Maas
4
S.M.
Schanberg,
and
5
M.D.
Armstrong,
6
E.K.
Gordon,
7
R.F.
Sugden
8
S.
Wilk
9
E. Watson,
Landis,
and
A.
J. Pharmacol.
K.
E.
Watson New
Black
S. Wilk G.R.
Chase,
Breese,
11
A.E.
Slaby
12
F. Karoum,
H. LeFevre,
13
F. Karoum.
J.C.
14
T.N.
Chase,
E.K.
15
G.V.
Erwin
New
York,
R.J.
E.K.
Wyatt
Gillin
in E.
E.
and
Gordon Usdin p. 161
Anal.
Gordon Bigelow
R.J.
and and
Wyatt,
L.K.Y. S.H.
163
(1968)
Biochem. 18
(1971)
(1963)
(1967)
I.J. Kopin, Biochim.
S.H.
10
157
373
493 147
Biochem.
Pharmacol.,
Biophys.
Acta.
Med.,
(1974)
11
25
17
(1957)
(1968)
247
422
32
2461
Snyder
(eds).
Frontiers
in
Catecholamine
Research.
p. 1067 Biochem., and
in Dementia L.B.
Ther..
Kopin,
and
Ther.,
and
Shaw,
I.J.
Usdin
1973.
and J. Roboz,
T.N.
EXP.
Breese
K.N.F.
and
Jr. J. Neurochem. EXP.
J. Neurochem..
in
York,
10
1973.
and
D. Eccleston.
Press,
and
.G.R.
McMiUan
J. Oliver,
Nashold,
J. Pharmacol.
J.J. Schidkraut,
and
and
Pergamon
B.S.
J. Jonason,
59
I.J. Kopin,
(1974)
in the Presenium, and
E.Costa,
Snyder
Charles
Clin.
J. Neurochem., Ng,
Chin
18
(1971)
C. Thomas,
Acta,
43
135
Springfield,
(1973)
Ill.,
1974
127
submitted
J. Neurochem.. (eds),
441
J. Neurochem,
Frontiers
21
(1973)
581
in Catecholamine
Research.
Pergamon
Press,