Normal excretion of m-hydroxymandelic acid in hypertensive patients

125

Clinica Chimica Acta, 109 (1981) 125-131 @ Elsevier/North-Holland Biomedical Press

CCA 1584

NORMAL EXCRETION OF m-HYDROXYMANDELIC HYPERTENSIVE PATIENTS

ACID IN

JAN R. CROWLEY, MARGARET W. COUCH, CLYDE M. WILLIAMS ROSE M. THREATTE and MELVIN J. FREGLY Veterans Administration Hospital and Departments of Radiology of Florida College of Medicine, Gainesville, FL 32610 (U.S.A.)

(Received

April llth,

*,

and Physiology,

University

1980)

Summary o-Hydroxymandelic acid (OHMA), m-hydroxymandelic acid (MHMA) and p-hydroxymandelic acid (PHMA) were measured in the urine of 42 normotensive and 54 hypertensive patients. Patients having high urinary MHMA levels were all found to be ingesting medications containing m-synephrine (phenylephrine). These patients also had high levels of urinary m-synephrine which was excreted as the glucuronide. When patients ingesting m-synephrine were excluded from the analysis, no significant differences were observed between the two groups for the urinary excretion of OHMA, MHMA and PHMA.

Introduction m-Hydroxymandelic acid (MHMA) has recently been identified in human urine by gas chromatography-mass spectrometry [ 11. It is probable that urinary MHMA originates from m-octopamine or m-synephrine or both. m-Octopamine has been identified in rat salivary gland [2], brain [ 31 and adrenal gland [4] and m-synephrine has been identified in bovine adrenal gland [5]. m-Octopamine has about l/100 the pressor activity of (-)-norepinephrine [6] but m-synephrine is a more powerful pressor agent having about l/3 the activity of norepinephrine [7]. While d,l-m-octopamine has about l/20 the pressor activity of Z-m-synephrine in vivo, it has about the same activity as m-synephrine in vitro using reactivity of aortic smooth muscle from rats as a measure of a-adrenergic activity [8]. Since both m-octopamine and m-synephrine have pressor

OHMA, o-hydroxymandelic acid; MHMA, m-hydroxymandelic acid; PHMA. p-hydroxymandelic acid: GC-MS-SIM, gas chromatography-mass spectrometry-selected ion monitoring. * Correspondence should be addressed to: Clyde M. Williams, M.D.. Department of Radiology, The J. Hillis Miller Health Center, University of Florida College of Medicine, Gainesville, FL 32610, U.S.A.

Abbreviations:

126

activity and since both occur role in the regulation of blood increased synthesis of one or pressure and in urinary MHMA

naturally, the possibility that they might play a pressure suggested to us the hypothesis that an both amines might lead to an increase in blood excretion.

Materials and methods Gas chromatography-mass

spectrometry-selected

ion monitoring

(GC-MS-SIM)

A Hewlett-Packard 5992A GC-MS was employed using a silanized glass column (1.8 m X 2 mm I.D.) packed with a 5% OV-101 on Chromosorb GHP 100/120 mesh (Supelco). A jet separator was used as the interface. The GC was operated isothermally. Chemicals

Standards were obtained from the following sources: MHMA and PHMA, Sigma Chemical; N-methyl-bis(trifluoroacetamide) (MBTFA), Pierce Chemical; m-synephrine (free base), Sterling Chemical. The piperazine salt of OHMA, 4,6[‘Hz]OHMA, 2,6[*Hz]PHMA and 2,4,6[*H,]MHMA were synthesized as described by Midgley et al. [l] and N-trideuteromethyl m-synephrine was synthesized as described by Midgley et al. [ 51. Methanolic HCl was prepared by the slow addition of 1 ml acetyl chloride to 4 ml dry methanol. Sample processing

procedure

and deriuatization

After assaying urine for creatinine, an internal standard containing 100 ng of 4,6[*H,]OHMA, 1000 ng of 2,4,6[2H,]MHMA and 20 000 ngof 2,6[?H,]PHMA was added to urine containing 10 mg creatinine. Acids were extracted and derivatized with pyridine-MBTFA (1 : 1, v/v) and methanolic HCl as described by Midgley et al. [ 11. The trifluoroacetoxy-methyl esters (TFA-ME) of OHMA, MHMA and PHMA were identified by retention time and the ratio of the characteristic ions m/z 315 and m/z 374. Quantitation was carried out by comparing the intensities of the ions m/z 315 and m/z 317 for OHMA-TFA-ME and PHMA-TFA-ME and of the ions m/z 315 and m/z 318 for MHMA-TFA-ME. Amines were extracted and derivatized with pyridine : MBTFA (1 : 1, v/v) as described by Midgley et al. [5]. The N-trifluoroacetyl-0-trifluoroacetoxy (TFA) derivative of m-synephrine was identified by retention time and the ratio of the intensities of the characteristic ions m/z 140 and m/z 455. Quantitative analysis was carried out by adding 1000 ng of N-trideuteromethyl m-synephrine to urine containing 10 mg creatinine a&comparing the ratio of the ions m/z 140 and m/z 143. Enzyme

hydrolysis

Enzymatic hydrolysis was carried out by adding 10 ml sodium acetate buffer (0.15 mol/l, pH 6.0) to urine containing 10 mg creatinine and incubating with (1) glucuronidase (Sigma Type H2, 120 000 Fishman units), or (2) sulfatase (Sigma Type H-l, 250 units) for 24 h at 37°C.

127

Patient selection

It is known that there are appreciable amounts of p-synephrine in orange juice (4.8 mg * 1-i) and orange meat (4 mg . g-1) and that p-synephrine is metabolized to PHMA [9,10] and we have confirmed that ingestion of orange juice leads to an increased urinary output of PHMA [ 11. A questionnaire was prepared to identify subjects who had ingested orange juice or orange meat during the previous 24 h and who had used any medication containing m-synephrine. All subjects described below filled out this questionnaire. Random samples of urine were obtained from students of the University of Florida who presented themselves to the student infirmary for a variety of complaints. Blood pressure (cuff method) was obtained in the sitting position and all patients with a systolic pressure of 150 mm Hg or more or a diastolic pressure of 90 mm Hg or more were excluded. Patients applying for general medical care to the out-patient facility of the Gainesville Veterans Administration Hospital receive a short history and physical examination which includes a measurement of blood pressure and a urinalysis. Over a period of 6 weeks urine samples were collected from patients over the age of 40 who were normotensive (systolic <150 mm Hg and diastolic <90 mm Hg) and hypertensive (systolic >150 mm Hg or diastolic >90 mm Hg). The condition under which the blood pressure was measured was the same as described above. Statistical analyses

Differences in group means were examined independent samples.

for differences

with the t-test for

Results Exogenous

m-synephrine

as a source

of urinary MHMA

The mean urinary excretion of MHMA in 10 normotensive adults was found to be 29 ng - mg-l creatinine [l]. In a preliminary analysis of urinary MHMA levels in hypertensive patients we found 3 patients with MHMA levels from 30-50 times this mean value. Review of the medical records in these 3 cases did not reveal any drug which might be expected to result in an increased MHMA excretion but on further questioning it was discovered that the patients had used nasal decongestants containing m-synephrine in the 24 h preceding the urinalysis. Two of three patients had used the m-synephrine containing medication daily for several years. In one patient the following experiment was carried out. Urine was divided into three aliquots: (1) the first aliquot was not treated; (2) the second aliquot was treated with glucuronidase; (3) the third ahquot was treated with sulfatase. The urine was then analyzed for m-synephrine. In the untreated urine and urine treated with sulfatase no m-synephrine could be detected. However, in the aliquot treated with glucuronidase large amounts of m-synephrine were detected. Fig. 1 shows a GC-MS-SIM analysis in which m-synephrine was identified and measured. The instrument monitored the base peak of endogenous m-synephrine-TFA (m/z 140) and of N-[‘H,]methyl-m-synephrine-TFA (m/z 143) and the respective molecular ions m/z 455 and m/z 458. The ratio of the ions m/z 143/m/z 458 of the internal standard

128

(85) was identical to the ratio of the ions m/z 140/m/z 455 of the endogenous amine. The amount in the urine was calculated from the ratio of the ions m/z 140/mlz 143. The three patients excreted m-synephrine as the glucuronide in amounts ranging from 500-1500 ng - mg-1 creatinine. In order to establish exogenous m-synephrine as a source of urinary MHMA, the following experiment was carried out: one of us (C.M.W.), on no medication, collected 24-h urine specimens without preservation for 8 days. Neosynephrine (0.570, Winthrop) was inhaled 4 times in each nostril, every 2 h from 0800-2200 h for 3 consecutive days. The total amount of liquid inhaled was 3.56 g containing 17.8 mg m-synephrine. Neither OHMA or PHMA excretion was affected by this drug. The results of the drug on MHMA and m-synephrine excretion are shown in Fig. 2. The normal excretion of MHMA is 29 ng - mg-l creatinine (range 11-71) [I]. This corresponds, assuming an average daily excretion of 1500 mg creatinine, to 43 p&day. The subject

m/z 143 (Full stole

Roti

411)

m/z m/z

5

85

455

m/r 140 (Full scale 152) 6

5

7

TimeCmin) Fig. 1. Four-ion GC-MS&M recording of TFA derivative of urinary amine extract after glucuronidase treatment from a patient who had inhaled a nasal decongestant containing msynephrine. 500 ng of N-trideuteromethyl m-synephrine was added to a sample of urine containing 1 mg creatinine. At an electron multiplier voltage of 2200 V, the characteristic base (m/r 140) and molecular ion (m/a 455) of endogenous m-synephrine dominate the tracing. Fig. 2. Urinary output of MHMA msynephrine over a 3-day period.

(0)

and m-synepbxine

(0)

glucuronide

during and after

inhalation

of

129

excreted 38 and 45 bg of MHMA on the two control days. Over the 4-day period after the beginning of m-synephrine inhalation, the subject excreted about 2.1 mg of MHMA above the control level of 42 yg/day. This amounts to about 11% of the amount inhaled. No free m-synephrine could be detected on any of the 8 days examined. About 5.6 mg of m-synephrine was excreted as the glucuronide over the 4-day period beginning with m-synephrine inhalation. This is about 31% of the amount inhaled. The Physician’s Desk Reference contains a list of 43 over-the-counter medications containing m-synephrine. Typical concentration for adult dosages were 5 mg in tablets and 5 mg/ml for nasal decongestant sprays. Urinary excretion

of OHMA,

MHMA

and PHMA in young

normotensive

adults

Subjects who had ingested orange juice or orange meat had elevated PHMA values while subjects who had ingested medications containing m-synephrine had elevated MHMA values. The ingestion of orange juice or orange meat had no effect on OHMA or MHMA excretion and the ingestion of medication containing m-synephrine had no effect on OHMA or PHMA excretion. The urinary excretion of the three hydroxymandelic acids of 30 males and 33 females who had not ingested orange juice was compared. The results of this analysis are shown in Table I. As expected the mean systolic and diastolic pressure of young females were significantly lower than young males. OHMA levels in young females were significantly higher (p < 0.05) than in young males and PHMA values were significantly lower (p < 0.025) in young females than in young males. Urinary MHMA values were not significantly different between the two groups. The magnitude of the OHMA difference was small and we have no explanation for it. The higher PHMA levels for males may have been due to unrecorded ingestion of p-synephrine in orange juice. Urinary excretion of OHMA, hypertensive patients

MHMA

and PHMA

in older

normotensive

and

MHMA values from patients who had ingested medication containing m-synePHMA values from patients who had phrine were excluded. In addition,

TABLE URINARY (ng

I EXCRETION

OF

OHMA.

MHMA

AND

PHMA

IN

NORMOTENSIVE

YOUNG

ADULTS

. m-1 creatine) Age

Systolic

(Yr)

(mm

22

128

14

12

12

Hg)

B.P.

Diastolic (mm

B.P.

MHMA

PHMA

6

19

1950

3

8

980

OHMA

Hg)

M&S mean S.D.

2

n

30

30

30

25

30

Fl?Wl&?S mean

23

105

69

9

25

1440

5

22

530

S.D. n P

5

30

14

9

33

33

33

18

32

31

NS



<0.05

NS

<0.025

130 TABLE

II

URINARY OLDER

EXCRETION MALES

. mg-l

(ng

OF

OHMA,

MHMA

AND

PHMA

IN NORMOTENSIVE

AND

HYPERTENSIVE

creatine)

Age

Systolic

(yr)

(mm

56

126

8

11

Hg)

B.P.

Diastolic

OHMA

MHMA

PHMA

76

10

53

3700

8

5

37

2200

(mm

B.P.

Hg)

Normotensive Mean S.D.

42

42

42

42

39

42

Mean

56

163

102

11

44

3500

S.D.

10

19

10

6

28

1700

n

54

54

54

47

54

NS



NS

NS

n

Hypertensiue

P

54

NS

ingested orange juice or orange meat were also excluded. The results are shown in Table II. The ages of the two groups were not significantly different. The systolic and diastolic pressures of the hypertensive group were significantly higher than in the normotensive group. The urinary OHMA, MHMA and PHMA values of the two groups were not significantly different. The OHMA, MHMA and PHMA levels of the normotensive older adults were significantly higher (JI < 0.005) than the levels of the normotensive younger adults. Discussion Exogenous m-synephrine when administered by nasal inhalation is excreted in the urine as m-synephrine glucuronide or as MHMA. However, not all of the exogenous m-synephrine can be accounted for as these two metabolites. About 11% is accounted for as MHMA and about 31% as phenylephrine glucuronide. There are several possible ways in which the remaining m-synephrine might be accounted for: (1) uptake and storage by storage vesicles similar to or identical with the ones which store norepinephrine; (2) conversion by tyrosine hydroxylase to epinephrine, and (3) conversion to other metabolites such as m-hydroxyphenylglycol or NJ-dimethyl-m-octopamine. Quantitative studies of the metabolism of exogenous m-octopamine have been carried out [ 111. During the 24 h after intravenous administration of tritiated m-octopamine about 60% was excreted as MHMA and about 20% as m-octopamine of which half was the free amine and half was conjugated. No quantitative studies of the metabolism of m-synephrine have been carried out. However, the metabolism of Etilephrine, the N-ethyl derivative of m-synephrine, has been studied by Hengstmann [ 121. During the 24 h after the administration of tritiated Etilephrine, approximately 4% was excreted in the urine as MHMA, and 72% as the amine of which about 213 was conjugated. The authors attributed this difference in part to the low affinity of monamine oxidase for N-substituted phenylalkylamines. These results suggest that urinary MHMA may originate primarily from endogenous m-octopamine and to a much lesser extent from endogenous m-synephrine. If this supposition is correct, our

131

findings of a normal excretion of MHMA in patients with hypertension does not exclude an increased synthesis of m-synephrine in that disorder. Furthermore, we did not measure free or conjugated m-synephrine in the urine of hypertensive patients. To establish the possible role of m-synephrine and m-octopamine and their conjugates in hypertension it will be necessary to develop a method for the direct determination of these amines in blood, tissue and urine. The GC-MS-SIM techniques we have employed using electron impact mass spectrometry can detect m-octopamine and m-synephrine down to levels of about 1 ng * ml-l or 1 ng * g-1. We have not been able to detect either m-octopamine or m-synephrine in blood or urine of normotensive humans at this level of sensitivity. Negative ion chemical ionization mass spectrometry under suitable conditions can detect amines down to the femtogram level. Work to explore the possibility of detecting these two amines by this technique is now in progress. References 1 Midgley. J.M., Couch, M.W.. Crowley. J.R. and Williams. C.M. (1979) Identification and quantitative determination of o- and m-hydroxymandelic acid in human urine. Biomed. Mass. Speck 6. 485-490 2 Robertson, H.A.. David, J.C. and Danielson, T.J. (1977) Effects of denervation on the levels of p-octopamine. m-octopamine (norphenylephrine) and phenylethanolamine in rat salivary gland, J. Neurochem. 29. 1137-1139 3 Danielson. T.J.. Boulton, A.A. and Robertson, H.A. (1977) m-Octopamine, p-octopamine and phenylethanolamine in rat brain: A sensitive, specific assay and the effects of some drugs. J. Neurochem. 29. 1131-1135 4 Williams, C.M. and Couch, M.W. (1978) Identification of ortho-octopamine and meta-octopamine in mammalian adrenal and salivary gland, Life Sci. 22. 2113-2120 5 Midgley. J.M., Couch. M.W., Crowley. J.R. and Williams. C.M. (1980) m-Synephrine: Normal occwrence in adrenal gland, J. Neurochem. 34,1225-1230 6 Fregly. M.J., Kelleher. D.L. and Williams, C.M. (1979) Adrenergic activity of ortho-, metoandparaoctopamine. Pharmacology 18.180-187 7 Pruss. T.P.. Maengwyn-Davies, G.D. and Wenzel. W. (1965) Comparison of effects of aromatic sympathomimetic amines on rabbit aortic strip and rabbit blood pressure, J. Pharmacol. Exp. Ther. 147, 76-85 8 Ress. R.J., Rahmani. M.A., Fregly, M.J.. Field, F.P. and Williams, C.M. (1980) Effects of isomers of octopamine on in vitro reactivity of vascular smooth muscle of rats. Pharmacology. in press 9 Gkssing. L. and Armstrong, M.D. (1963) Occurrence of (-_)-sympatol in oranges. Proc. Sot. Exp. Biol. Med. 114,226-229 10 Stewart. I., Newhall, W.F. and Edwards, G.J. (1964) The isolation and identification of 1-synephrine in the leaves and fruit of citrus, J. Biol. Chem. 239, 930-932 11 Hengstmann. J.H.. Konen, W., Konen. C., Eichelbaum, M. and Den&x. H.J. (1975) Bioavailability of m-octopamine in man related to its metabolism. Eur. J. Clin. Pharmacol. 8. 33-39 12 Hengstmann. J.H., Weyand. V. and Dengler. H.J. (1975) The physiological disposition of Etilefrine in man, Eur. J. Clin:Pharmacol. 9.179-187