Effects of large variations in diet on free catecholamines and their metabolites in urine

J. psvchiat.

Res.,

1970,

Vol. 7,

pp. 263-273.

Pergamon

Press. Printed in Great Britain.

EFFECTS OF LARGE VARIATIONS IN DIET ON FREE CATECHOLAMINES AND THEIR METABOLITES IN URINE P. V. CARDON and F. G. GUGGENHEIM* Laboratory of Clinical Science, National Institute of Mental Health, U.S. Department of Health, Education and Welfare, Bethesda, Maryland 25 August

1969)

25 November

1969)

(Received (Revised

IN THE past decade there has been extensive

study of catecholamines and their metabolites in urine. Interpretation of data obtained in clinical studies would be facilitated by additional quantitative information regarding the effect of variations in diet on estimates of urinary excretion of the substances studied. From most points of view it is usually sufficient to know, for example, that according to one current specific method, dietary 3-hydroxy-4-methoxy-phenyl-hydracrylic acid (present in coffee) makes chromatographic separation of VMA more difficult;1 or that bananas and, to a lesser degree many other fruits and vegetables, contain norepinephrine and dopamine.%3 Error can theoretically be minimized in the short run at least by appropriate exclusions from the diet and that is often the best expedient. In the study of psychiatric patients over periods of weeks or months the matter is less simple. Prolonged restriction of diet might seriously impair morale and conceivably even general health, particularly when anorexia is present. Accidental or deliberate lapses from a dietary regimen require realistic and appropriate responses from physicians and nurses within the therapeutical clinical setting, as well as evaluation of their probable impact on laboratory results. Days spent away from a hospital are often therapeutically desirable and rich in important events in patients’ lives, but control of diet at those times is difficult. Intermittent diet restriction, whether according to a preset schedule or in anticipation of an expected clinical change, has obvious disadvantages. The present study was prompted by lack of adequate systematic data on the effect of diet on the excretion of catecholamines and their metabolites. It addresses itself to the general question: How do the effects of variation in diet compare in magnitude to the variations observed among or within individuals which are independent of diet? METHODS Seven male volunteer college students living at the Clincal Center of the National Institutes of Health served as subjects. They were in good general health. Age range was 18-21 years; median 19 years. Body weight range was 50-75 kg; median 66 kg. Psychiatric * present address : Massachusetts 02114.

General Hospital

263

Department

of Psychiatry, Boston, Massachusetts

264

P. V. CARWN AND F. G. GUGGENHEIM

consultation (obtained routinely at the time of admission) noted that 3 of the 7 subjects (subjects 1,4 and 5) gave histories of difficulties in social and academic adjustment sufficient to justify diagnosis of ‘adjustment reaction of adolescence’. All the subjects were encouraged to report symptoms of every type to the staff throughout their hospital stay in order to identify immediately any condition which might contraindicate a research procedure. In this setting of deliberately fostered over-reporting of symptoms, few psychiatric symptoms were reported and these were considered by subjects and staff to be quite ordinary. The subjects spent most of each week in sedentary scientific educational activity. Physical exercise was not formally controlled, but on the days of urine collection it was neither strenuous nor sustained. Subjects 1, 3 and 4 smoked cigarettes ad libitum. The subjects were requested to exercise and smoke in similar amounts on all urine collection days. Three diets were employed. ‘Diet A’ contained in excess foods ordinarily excluded from diets intended to minimize error. Included daily were: 2-4 glasses of orange juice, 3-6 cups of coffee or tea, l-3 cola drinks, a 40-g bar of chocolate, 4-6 servings of fruit, 4-6 servings of vegetables, 150 g of potatoes, l-2 bananas, and at least 1 portion of dessert containing vanilla. Almost all subjects on this diet said they were getting too much to eat. ‘Diet B’ was a moderately restrictive diet which might be easily tolerated by patients for long periods. Although it contained food that might add to the apparent or real excretion of catecholamines and their metabolites, these substances (fruit, vegetables, coffee, dessert) were given each day in moderate and relatively constant amounts. Included daily were: 1 glass of orange juice, 1 cup of coffee, 3 servings of vegetables, 150 g of potatoes, 3 servings of fruit (no bananas), and 1 portion of dessert containing vanilla. ‘Diet C’ was a severely restricted acid-ash diet employed in one of the NIMH research units. Excluded foods were: cola and alcoholic beverages; desserts containing vanilla; fruits except weak lemonade; all vegetables except corn, rice, noodles, and spaghetti; coffee, tea and chocolate; more than 1 teaspoon of jelly/meal; milk; foods with artificial flavoring or coloring. Average daily protein intake on the A, B and C diets was calculated in retrospect to be 89.5, 102.6 and 96.0 g, respectively. The 3 diets were given consecutively in a Latin Square design, each diet for one week. Because 7 subjects were available, but only 6 possible diet sequences, 2 subjects were assigned sequence CBA. Each diet began on a Friday morning. Urine collection was begun at 8 a.m. the following Tuesday, and three consecutive 24-hr urine collections were then made. Sodium metabisulfite or dilute hydrochloric acid were added as preservatives. Urine was refrigerated as it was being collected. Total 24-hr volume was then measured, and an aliquot was taken, frozen, and subsequently analyzed in duplicate. Thus, determinations of each substance studied were made for 63 subject days. Analytic procedures were as follows. Creatinine: a modification of the Jaffee reaction.4 Free epinephrine (E) and free norepinephrine (NE): extraction by the method of ANTON and SAYRE.~Fluorimetric determination by a modification of the trihydroxyindole procedure -differential oxidation with ferricyanide at pH 7.0 and pH 3.5 using ZnCls and ethylenediamine. Metanephrine (M) and normetanephrine (NM): the method of GORDON et al. which has been described elsewhere.6 VMA: the method of WEISE et a1.l Free dopamine: a modification of the method of MCGEER and MCGEER.’

EFFECTS OF LARGEVARIATIONSIN DIET ON FREECATECHVLAMINES AND

THEIRMETABOLITES IN UIUNE 265

RESULTS

Although much care was taken to insure complete and accurate urine collections, and no urine loss is known to have occurred, there was much variation in the estimated daily creatinine excretion of most subjects. The range of coefficients of variation within subjects was lo-26 per cent, median 16 per cent. For this reason the data that follow usually are expressed in relation to creatinine excretion (‘creatinine correction’), with explicit reference to 24-hr values when these are presented. Diet efsects on average values See Table 1 and Fig. 1. Average estimated than on B or C diets; this is of questionable TABLE 1.

Creatinine (mg/24 hr) D (pgjg Creatinine) NE (pg/g Creatinine) E&g/g Creatinine) NM (pg/g Creatinine) M&g/g Creatinine) VMA (mg/g Creatinine)

creatinine excretion was lower on the A diet statistical significance (see below). Putting

AVERAGERATESOF EXCRETION/gCREATININE

A

Diets B

C

1667 184.6 25.2 18.8 228.0 104.5 2.22

1873 173.1 24.4 16.9 180.4 104.1 2.08

1849 164.7 23.7 13.6 177.9 95.2 1.73

F (Diets) 3.63 0.55 0.15 1.79 13.121 2.66 4.06

F (Subjects) 9.581 5.32t 11.61 3.33* 10.61 30.5: 19.61

* p less than 0.05. t p less than 0.01. $p less than 0031. Average values for free dopamine (D), free norepinephrine (NE), free epinephrine (E), normetanephrine (NM), metanephrine (M), and 3-methoxy-4-hydroxymandelic acid (VMA) excreted per gram of creatinine. ‘F’ ratios derived from two-way analysis of variance: diet or subject mean square divided by diet subject interaction mean square.

aside the question of statistical significance, the effect of the creatinine correction thus is upward bias of the A diet averages for the other substances. Whether this makes the A diet data more valid or less valid is discussed later. Average values per g of creatinine of the amines and metabolites measured were highest on the A diet and lowest on the C diet. Two-way analysis of variance is an appropriate statistical treatment, but independence of sequential samples and homogeneity of variance among individuals cannot be assumed. The denominators of the F ratios in Table 1 are the diet X subjects interaction terms. This gives the same F ratios as would two-way analysis of the averages of the three values for each subject on each diet (N = 21). Degrees of freedom are assumed to be 2 and 12 for diets. By this evaluation, there was a highly significant diet effect on NM (p tO.OOl), a significant effect on VMA (p
P. V. CARDON AND F. G. GUGGENHEIM

266

4O-rlOO

&~rn CR

20

0

1

0

SUBJECTS

. ...

.

.. ..

l

l

. .

IO

II

1

pgmM/gm I50 IO CR

.

I

-

I

2

3

4

5

6

7

FIG. 1. Average values (n = 3) for each subject on each diet. Coefficients of variation (closed circles) are plotted on a logarithmic scale. They are included as a simple visual indicator of the magnitude and consistency of the effects of diet on day-to-day variation.

EFFECTS OFLARGEVARIATIONS IN DIETON FREECATECHOLAMINES ANDTHEIRMETABOLITES IN URINE 267

diet effects. A followup study of the effects of one of the dietary variables, caffeine, on E and A4 will be reported elsewhere.8 The averages and F ratios derived from similar analysis of the quantities excreted per 24 hr appear in Table 2. A significant effect of diet on A4 is present (p
Creatinine (mg/24 hr) D @g/24 hr NE &g/24 hr) E (pgi24 hr) NM &g/24 hr) M (pgI24 hr) VMA (mg/24 hr)

AVERAGERATESOF EXCRETION/DAY

A

Diets B

c

1667 291.8 40.3 29.4 366.5 175.8 3.82

1873 341.1 44.5 30.0 337.5 197.7 4.05

1849 394.5 43.1 24.0 328.5 175.1 3.37

F (Diets) 3.63 0.30 0.85 1.29 0.72 7.03t 1.13

F (Subjects) 9.583 0.90 8.03-t 2.54 6.54t 70.13$ 15.81

* p less than 0.05. t p less than 0.01. $ p less than OXlOl. Same as Table 1, but giving amounts excreted in 24 hr.

to interpret because in this case the B diet average not only is significantly higher than the C diet average, where creatinine excretion is comparable, but also significantly higher than the A diet average-presumably because of the lower creatinine values. Diet effects in the 24-hr data for other substances do not approach significance. Diet and intra-subject variance Because the C diet was the most constant in catecholamine content, one would expect any effects of the more liberal diets to be reflected in increased variability. Table 3 is directed to this question. One-way analysis of variance was done for each diet, and from the error mean squares term a ‘coefficient of variation’ (‘CV’) was computed. ‘CV’ = 100 x EMSlP x diet grand mean-l. These appear as the numbers in parentheses in Table 3. Variance of NM and E appear to be most affected by liberalization of the diet and NE least. Fig. 1 offers another approach to the question, indicating the extent to which each subject exhibited these average trends. The comparison of B and C diets is of the greatest practical interest. It can be seen that except for E, variance is smaller on the B diet as often as it is larger. Variance and creatinine correction Table 3 also shows how the ‘coefficients of variation’ of 24-hr values were changed by the creatinine correction. This creatinine correction, when applied to data for each of the diets, consistently reduced ‘CV’ for NM, M, and VMA, but not for the free amines, D, NE, and E. Intersubject variance Tables 1 and 2 give the over-all F ratios of intersubject variance to intrasubject variance. As in the evaluation of diet effects, the denominators of the F ratios are the diet subject

P.V. CARDONAND

268

TABLE3.

(per g Cr)

GUGGENHEIM

DIET ANDINTRA-SUBJECT VARIANCE

A Diet Coefficients of variation (Subccts) (per cent) D

F.G.

B Diet

(SubyLs)

Coefficients of variation (per cent)

C Diet Coefficients of variation (SubyLcts) (per cent)

5.58t 4.04*

(24.0) (19.4)

2.55 1.43

(39.7) (42.3)

5.30t 1.52

(17.7) (22.5)

6.29.t 8.07f

(24.0) (19.1)

4.01* 4.70t

(25.7) (20.1)

6.70t 4.78t

(24.3) (25.7)

(per 24 hr)

2.91* 4.617

(41.6) (28.8)

4.27* 2.14

(34.7) (36.4)

10.16f 7.751

(21.4) (21.2)

NM (per g Cr) (per 24 hr)

1.82 2.23

(26.2) (28.6)

2.82 5.29t

(18.3) (20.4)

10.43$ 19.54%

(14.0) (15.2)

M (per g Cr) (per 24 hr)

7.11.t 5.21t

(18.1) (26.1)

12.063 3.32

(13.9) (32.3)

10.74$ 5.957

(14.5) (24.2)

10.75: 7.79$

(21.8) (30.9)

5.83t 4.67t

(33.7) (46.7)

12.308 15.02$

(20.8) (26.0)

0.76

(21.0)

2.8*

(17.8)

4.55t

(16.2)

(per 24 hr) NE (per g Cr)

(per 24 hr) E (per g Cr)

VMA (per g Cr) (per 24 hr) Cr (per 24 hr) * p less than 0.05. t p less than 0.01. $ p less than 0.001.

Effect of diet on intrasubject

variance. Results of one-way analyses of variance for each diet.

‘F Subjects’: subject mean square divided by error mean square, d f 6, 14. ‘Coefficient of variation’: 100 x (error mean square)‘/’ x diet grand mean-l.

interaction mean squares. Except for E and D they are highly significant. Average values for NE and for all metabolites discriminate as reliably among subjects as do the creatinine values. Table 3 lists the F ratios derived from one-way analyses of variance for each diet. Predictably (from the given information about intrasubject variance) liberalizing the diet reduces markedly the confidence with which one can distinguish among subjects with regard to excretion rates of E and NM. By contrast, analogous effects involving VMA, NE, D, and M must be slight if they do in fact exist. It appears that with control of diet appropriate to the substance measured, as few as three urine samples may be sufficient to define for many purposes an individual’s usual excretion rate of all the substances measured. Whether similar reliability in normal subjects would emerge from three samples spaced over months or years is an open question. Relative importance of diet variance and inter-subject variance It is clear that the diet has larger effects on some substances than on others, both on average values and on variance, and that intersubject differences are generally more striking than the diet effects. However, some of the practical conclusions remain implicit and perhaps

EFFECTSOFLARGEVARIATIONS IN DIETON FREECATECHOLAMINES AND THEIRMETABOLITES IN URINE 269

obscure. Table 4 is presented to bring both the practical implications and the limitations of the data into sharper focus. The standard deviations of the C diet values from their common means have been computed as estimates of the range of values which might be observed in a group of subjects when diet is closely restricted. The differences between A or B diet grand means and C diet grand means are given as standard scores (fractions of the C diet standard deviations). These may be viewed as estimates of how much a subject would be TABLE4.

DIET AND

INTER-SUBJECT VARIANCE

AZ B-C D @g/g Creatinine)

0.19 0.07 0.59 0.05 0.32 0.47

NE @g/g Creatinine) E @g/g Creatinine) NM (pg/g Creatinine)

A4 @g/g Creatinine) T/MA (mg/g Creatinine) Per cent of population Band width at mean (Z)

33 0.89

25 0.65

UL 90 per cent band AZ A-C 1.09 0.51 1.16 0.73 0.72 1.09 20 0.49

0.45 0.16 0.93 1.03 0.34 0.65 12.5 0.31

10 0.24

UL 90 per cent band 1.45 0.66 1.91 1.49 0.48 1.07 5 0.10

Comparison of diet effects and intersubject variance. Differences between average C diet values and average A and B diet values are expressed as standard deviations of the C diet distribution (‘AZ’). ‘UL 90 per cent band’ : AZ + 2 SEM of AZ, n = 6. The lowest two lines, taken from a standard table of the distribution of the normal probability integral, are given so that the changes observed with changes in diet may be approximately related to the ranges of C diet values exhibited by the seven subjects. For example, if urinary excretion rates of NE by 100 subjects on the C diet were found to be distributed normally within the range found in the present study, an individual exhibiting an approximately average excretion rate of NE while on the C diet, and exhibiting the observed meandZ of 0.16 when placed on the A diet, would change his rank order by less than 10. However, the standard error of the mean AZ is large, and there is roughly one chance in ten that the mean change in rank order might be as large as 20 (UL 90 per cent band is 0.51, band width at mean for 20 per cent of population is 0.49).

displaced from his ‘true’ place in the group distribution if he were on one diet and all the others were on another diet. The same statistic is of course obtained if each subject’s average ‘displacement’ is calculated separately and the mean of these is then calculated. This was actually done, and the standard errors of the means were computed in order to give confidence limits for the displacement estimates. The upper limits of the 90 per cent confidence bands (+2 SEM for n = 6) are given. Finally, from the distribution of the normal probability integral, the band widths (or standard score ‘displacements’), which include (or ‘pass over’) various fractions of the normal distribution at its mean, are given at the bottom of the table. None of the average B-C displacements exceeds one quartile. Only the average A-C displacements for E and NM exceed one quartile. Only in the case of NE can it be said with 90 per cent confidence that no diet effects would result in a shift of more than one quartile in a group of subjects exhibiting this range of NE excretion rates.

270

P. V. CARDON AND F. G. GUGGENHEIM DISCUSSION

The interpretation of the creatinine data will be discussed first. A frequent reason for measuring urinary catecholamines and metabolites is to assess indirectly the level of activity of noradrenergic nerves and adrenal medulla. It is assumed that level of activity is roughly reflected in plasma concentrations of the amines and/or their metabolites, and that the rates of their renal excretion are in turn indices of plasma concentrations. In such a context it is desirable to make some sort of correction for collection errors and variations in renal clearance. In the absence of evidence that there is net tubular secretion of the catecholamines and metabolites (in mammals),9 the creatinine correction can be expected to reduce collection error and error due to variations in renal clearance if the following assumptions are also valid : (1) The rate of creatinine formation is constant in each individual. (2) In each individual a constant fraction of the creatinine formed is excreted in the urine. (3) A negligible fraction of the creatinine excreted is exogenous. (4) Laboratory error in creatinine measurement is negligible. In the present study it is safe to assume that the last two of the four conditions listed above were met. Differences in cooked protein intake of 1 g/kg are reported to change creatinine excretion by approximately 100 mg/day .I0 In the present study the difference in average daily protein intake between the A and B diets was approximately 0.2 g/kg, and the probable effect of this difference on creatinine excretion is approximately 20 mg/day, which is negligible. The creatinine determinations were done in duplicate by a simple and accurate procedure. Furthermore, the study design is such that any systematic batch-to-batch errors in creatinine determination would affect the three diet averages equally. Constancy of the rate of creatinine formation is assumed in most textbooks and mongraphs which deal with the subject. The main evidence for this, starting with the work of FOLIN,~~ is that urinary excretion rate is usually constant. However, it is the experience of this laboratory that wide variation in creatinine excretion is not uncommon in normal volunteers and in psychiatric patients at times when we believe that urine collections are complete. Repeated independent measurements of rate of creatinine formation have not been made in individuals who, like some of the subjects in the present study and in others,la-15 exhibit for some reason or combination of reasons large variation in estimated excretion rate. The prevalent assumption that virtually all of the creatinine formed is always excreted in the urine, appears also to rest on inadequate data and in part on circular reasoning.16 Returning to the present study, it is not impossible that rate of creatinine formation or the fraction excreted was in fact less when subjects were on the A diet-either by chance or as the result of some obscure effect of the A diet constituents. If this were so, we would conclude that large variations in diet have very little or no effect on urinary excretion of the catecholamines or their metabolites (Table 2). The more conventional interpretation of the creatinine data, and in our opinion probably the correct interpretation, is that the lower average creatinine excretion estimate made for the A diet days is due mainly to chance clustering of instances of poor collection and/or reduced glomeruler filtration rate. If that is so, we conclude that the creatinine correction reduced these errors sufficiently to reveal true effects of diet on NM and VMA, and to

EFFECTSOF LARGE VARIATIONSIN DIET ON FREECATECHOLAMINES AND THEIR METABOLITES IN URINE 271

generate the hypothesis, subsequently confirmed by the present authors* and also independently,17 that coffee stimulates secretion of E. It was predictable that NM and VMA would be most affected by the diets employed. The principal diet change was in content of NE and D, but it was expected that these amines would be mostly conjugated, oxidized, or methylated in the liver following absorption from the gut. The present study shows that over a wide range of amine ingestion in food, not enough free amines reach the systemic circulation to measurably change rate of free amine excretion. Total urinary D and NE (free plus conjugated) would undoubtedly have been found to be affected if they had been measured.18 The progressive increase in VMA with more liberal diets is presumed to result from increase in NE ingestion. Assuming the validity of the creatinine correction, the change from the C to the B’diet increased daily VA4A excretion by approximately 0.7 mg, and the addition of more fruits, vegetables, and bananas further increased VMA excretion by approximately 0.3 mg. (It will be noted that in the case of NM, by far the larger increment in NM excretion occurs between the B and A diets, which is more in keeping with the larger increment in NE content of the diet.) The A-C difference in VMA excretion of approximately 1 mg is roughly consistent with published data. Ingestion of four bananas has increased VMA excretion by 0.6 mg.2 Because the isotopic tracer method employed is specific for VMA, other phenolic acids contained in the more liberal diets (particularly in coffee and vanilla) should not have changed average values, but may well have decreased the precision of measurement. Any increase in endogenous E secretion would increase VMA excretion only by approximately as much as the increase in M excretion.19 In the present study this increase would have been slight-of the order of 20-30 r_lg. If there were real but statistically insignificant diet effects on urinary E and M, they must have resulted from changes in rate of E secretion. Food has not been shown to contain E or M. Furthermore, exogenous E would be metabolized like exogenous NE and would not appear as free urinary E. As to how much control of diet is necessary in clinical studies, obviously this depends on the study design and what is measured. Urinary free NE is unique in that diet seems to affect neither quantity nor variability. Free D probably may be classed with NE. As for free E, avoidance of caffeine would appear to be the only necessary diet control. The greater variability in E noted when a ‘constant’ amount of coffee was taken each day (diet B), suggests that a ‘constant’ coffee intake may not only increase average values, but also introduce additional ‘random’ variability. The situation may be different for M. A ‘constant’ coffee intake might increase average values appreciably, but such an increase would be small relative to intersubject variance, and intra-subject variance appears to be affected less than it is in the case of E. Studies of urinary NM and VMA require closer attention to diet. Provided that the chemical analyses are specific, the NE content of the diet is the most important factor. Control of caffeine is unnecessary because it does not affect urinary NM and VMA.8 Longitudinal studies where each subject is his own control would be most vulnerable to day-to-day fluctuations in dietary NE. Dietary control of NE would be particularly important if the intent of the studies were to follow daily NM or VMA excretion in relation to other variables measured day by day. On the other hand, if major changes in clinical state occurred

272

P. V. CARDON

AND F. G. GUGGENHEIM

over time, as in comparing ‘ill’ and ‘recovered’ periods, or periods on and off medication, and if the subjects didn’t grossly change their eating of fruits and vegetables, analysis of more urine samples in each period or pooling of samples might be preferable to rigid control of diet. SUMMARY

Urinary excretion of free catecholamines and their major metabolites was studied in seven normal young men while they were on three diets which were excessive, moderately restricted, or very low in catecholamine and caffeine content. When results are expressed in relation to urinary creatinine, the most liberal diet increased normetanephrine (NM) and P’MA excretion by about 30 per cent. Free epinephrine (E) and metanephrine (M) excretion rates also increased by 40 per cent and 10 per cent, respectively, but the changes were not statistically significant. Changes in free norepinephrine (NE) and free dopamine (D) excretion were small and did not approach statistical significance. When results are expressed as quantities excreted per day, the only statistically significant difference associated with diet was greater excretion of M during the moderately restricted diet. Intra-subject variation in estimated excretion of creatinine was large, with a median coefficient of variation of 16 per cent. When amines and metabolites were expressed per unit of creatinine, intrasubject variance of metabolites was made smaller but variance of free amines was not. This ‘creatinine correction’ tended to enhance the statistical significance of intersubject differences. The more liberal diets tended to increase intrasubject variance, particularly of E and NM. Variance of NE was least affected. Except for E and NM, where it was roughly equal to diet effects, intersubject variance was much greater than the diet effects. It is concluded that in studies of urinary NM and VMA, attention must be given to the NE content of the diet. In studies involving E and M, caffeine should be proscribed or controlled. In the case of free NE, and probably of free D, no control appears to be necessary.

Acknowledgements-The authors are indebted to EDNA GORDON and JERRY OLIVER for the chemical determinations, and to GREY B. THOMPSONfor her computer analyses of the data.

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7.

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