INTRODUCTION

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PROTEIN INGESTION LEADS to increased sodium, water and urinary free dopamine (free refers to unconjugated dopamine and is unrelated to its protein binding) excretion in rats (14) and humans (27), the human response being attenuated with progressing age (9). The dopamine-mediated natriuretic response to protein and its blood pressure-lowering effect may play a role in the inverse relationship between dietary protein markers and blood pressure in the population-based Intersalt study (22). Hypertensive subjects, compared with normotensive subjects, were found to have a decreased urinary free dopamine response in the postabsorptive state after protein ingestion (4). All these studies were based on urinary free dopamine excretion and evaluated the precursor role of 3,4-dihydroxyphenylalanine (DOPA) in the dopamine generation by using carbidopa, an aromatic acid decarboxylase inhibitor. This approach suggested that extraneuronal DOPA decarboxylation to dopamine contributes to the protein-induced natriuresis (27).

The present study extended the protein-induced paradigm in the following directions. 1) The study directly determined the catecholamine synthesis and metabolism cascade from plasma tyrosine through plasma and urinary DOPA and its metabolites (DOPA sulfate, 3-O-methyl-DOPA); dopamine, norepinephrine (NE), epinephrine (Epi), and their sulfoconjugates; the dopamine metabolites homovannilic acid (HVA) and dopacetic acid (DOPAC); and other homeostatic responses, such as plasma renin activity (PRA), aldosterone, and atrial natriuretic factor (ANF), all in relationship to changes in natriuresis and cardiovascular indexes. 2) It paid equal attention to the respective plasma catecholamines as studies previously focusing on urinary catecholamines. 3) Changes were in shorter collection periods in more numerous than previously reported age-matched cohorts of normotensive (27) and hypertensive (4) subjects kept on a comparable baseline salt balance state.

This study indicates that protein-induced plasma free dopamine increase relates better than urinary free dopamine increase to the postprandial natriuresis. A failure to increase plasma free dopamine in hypertension discriminates the dopamine generation defect in hypertension (4) as demonstrated also by oral DOPA administration (16). It confirms in humans a previous observation in rats (14) that short-term responses to protein ingestion affect dopamine in parallel with Epi. Thus this study represents a differential catecholamine response to protein intake affecting jointly the autocrine-paracrine dopamine (11) and Epi-represented adrenomedullary systems distinct from the sympathetic terminals reflected by NE. Finally, circulating sulfoconjugates of DOPA and Epi increased absolutely in this study after protein ingestion as did levels of dopamine sulfate relative to free dopamine; this may have contributed to some of the responses of hypertensive subjects distinguishing them from control subjects.

	METHODS

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Twenty-two control subjects in good health (17 men and 5 women; mean age 49.6 yr) and 18 age-matched control hypertensive patients (14 men and 4 women; mean age 49.3 yr) of comparable weight (74.9 ± 2.1 vs. 71.3 ± 2 kg) were studied. The Ethics Committee approved the protocol and informed consent was received from all subjects. All subjects were Caucasians and nonsmokers; none of the women had taken oral contraceptives or hormones. Drug abuse was eliminated by urinary screening.

All patients had been mildly to moderately hypertensive for 5-15 years (defined by blood pressure without medication >145/90 mmHg but never higher, 3 days off medication, than 190/105 mmHg). Their fundi changes never exceeded grade I. Eight of the patients had electrocardiogram or cardiac ultrasound changes compatible with left ventricular hypertrophy, and there was never more than trace proteinuria and a very moderate decrease in the creatinine clearance, although still within normal for age-corrected limits.

Before the protocol was initiated, all subjects had a complete workup and were found to have a normal biochemical profile, blood count, urine analysis, and negative hepatitis and human immunodeficiency serology. In hypertensive patients all drug treatment was stopped 1 wk before the study; none used long-acting drugs.

Three days before the study, all subjects were placed on a diet containing 150 mmol Na⁺, 100 mmol K⁺, and ~90 g protein. Before the protocol, a 24-h urine sample was collected to establish a comparable state of Na⁺ excretion in both groups. Each subject underwent two different randomly chosen meal studies, one of tuna and another of an equivalent electrolyte broth, separated by an interval of at least 10 days. Subjects started the test morning after an overnight fast. They remained supine during the study but were permitted to stand to void to aid in complete bladder emptying. An indwelling venous catheter was inserted and 30 min after, 60 min and time 0 blood samples were obtained. Urine samples were obtained hourly starting 60 min to time 0 followed by five hourly urinary samples. To ensure sufficient urine volume, all subjects received an oral water load of 500 ml between 60 min and time 0 and 200 ml each following hour. After time 0, five hourly blood samples were drawn from the catheter. Blood pressure and pulse rates were measured each hour.

At time 0, the test meal consisted either of chunk light tuna in pure distilled water (1 g/kg wt; Chicken of the Sea, Van Camp Seafood, St. Louis, MO) containing 40 g protein per container (net wt 6 1/8 oz) or an electrolyte equivalent noncaloric meal in the form of broth. Both meals contained a mean of 3 mmol Na⁺ and, dependent on the weight of the patient, between 12 and 18 mmol K⁺. The determined approximate mean free DOPA content ingested with tuna was low, ~0.3 µg.

Blood samples were drawn at times 60, 0, 60, 120, 180, 240, 300 min for tyrosine; 3-O-methyl-DOPA; DOPA and its sulfate; NE, Epi, dopamine, and their sulfates; as well as Na⁺, K⁺ creatinine, uric acid, urea, plasma renin activity (PRA), aldosterone, and atrial natriuretic factor (ANF) into chilled tubes containing EDTA. Blood samples were immediately centrifuged at 5,000 g at 4°C for 15 min, and plasma was frozen at 70°C until assayed. The 24-h urine collected preceding the test and aliquots of each hourly sample were placed into separate containers for electrolyte, creatinine, dopamine, NE, Epi, DOPA, and their sulfates, as well as 3-O-methyl DOPA, HVA, and DOPAC. Electrolytes and creatinine were immediately assayed, and all other urinary samples were frozen at 70°C until assay.

Free and sulfoconjugated catecholamines (NE, Epi, dopamine) in plasma and urine were determined radioenzymatically using catechol-ortho-methyl-transferase before and after sulfatase hydrolysis (25). Plasma and urinary 3-O-methyl- DOPA and DOPA, as well as DOPA sulfate (6, 13), DOPAC, HVA (26), and plasma tyrosine (7) were measured by HPLC with electrochemical detection. The inter- and intra-assay variability of these methods was between 7 and 12% and 4 and 8%, respectively. The detection limits for NE, Epi, and dopamine were 0.11 pmol/ml. Serum and urinary Na⁺ and K⁺ were measured by flame photometry, creatinine, urea, and albumin by automatic multichannel standard multiple analyser measurements. Creatinine clearance corrected for body surface was calculated from urinary and plasma creatinine concentrations and urinary volume. PRA, aldosterone (19), and ANF (18) were determined by radioimmunoassay.

Data analysis. For a given characteristic (e.g., systolic pressure), for each of two experimental conditions (ingestion of tuna and control ingestion of broth), and for each of two groups (A = control and B = hypertensive), six determinations were available, e.g., baseline (A1 or B1) and after 1, 2, 3, 4, and 5 h (A2-6 or B2-6). These observations were transformed into percentage, i.e., A1 or B1 was replaced by 100% and the remaining by percent of the baseline. A repeated-measures analysis of variance on the logarithms of these percentages was performed using the general linear models procedure of the SAS software. The logs were taken in order to stabilize the variances. When the profile analysis showed a significant variation in time, the successive differences in time were examined (e.g., hour 1 was compared with hours 2, 3, 4, and 5). For several variables, homogeneity of groups A and B was examined by comparing the differences in experimental conditions for each subject, i.e., the difference between ingestion of tuna and control was computed for every subject under each group and these differences were compared by means of a two-sample t-test. Furthermore, homogeneity of the experimental conditions under each group was examined by performing one-sample t-tests based on the differences of both groups. For some of these variables, the two-sample t-test was accompanied by some nonparametric tests, namely, the Wilcoxon-Mann-Whitney test, the median test, and the van der Waarden-normal scores test. A correlation analysis of original values between several variables and/or functions of variables was performed using the Corr procedure of the SAS software, which provides Pearson and Spearman correlation coefficients.

	RESULTS

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The baseline values preceding protein intake or the control period of some overall indexes were (Table 1) without difference between the preprotein and precontrol period values, as well as between control and hypertensive subjects, except for higher systolic and diastolic blood pressure in the hypertensive group. The 1-5 h after protein intake or control period percent changes were compared with the baseline (100%). Protein intake had no effect on systolic or diastolic blood pressure in controls but resulted, in hypertensive subjects, in significant decreases in both pressures over 2 h, becoming lower than in the control period without protein 3 h after protein ingestion but not lower than in control subjects. The protein-induced systolic blood pressure decrease in hypertensive subjects became significantly different from controls 2, 3, and 4 h after protein. Pulse rate tended to initially exhibit bed rest-related decrease. This was followed in control subjects by a significant increase in pulse rate 3 h after protein intake but had no effect on pulse rates after protein in hypertensive subjects or any control period without protein. Plasma urea progressively increased in both groups after protein (significantly more in hypertensive subjects 2-5 h postprandially) but not in the control period. Urinary volume increase became higher after protein intake than in the control period in control, but not in hypertensive, subjects, with an intergroup difference becoming significant 4-5 h after protein intake. After protein intake plasma albumin did not change in control subjects, but a tendency toward a postprotein decrease in hypertensive subjects between 2 and 5 h after protein intake made them significantly different from controls.

Protein intake-induced plasma changes (Fig. 1) from baseline values consisted of significant free plasma DOPA increase 2 h after protein intake in control subjects but no change in hypertensives. Opposite this, plasma DOPA sulfate increased in hypertensives but did not change after protein intake in controls, a difference becoming significant 2 h postprotein. Plasma free dopamine progressively increased in controls after protein intake but remained unchanged in hypertensives, resulting in 2, 4, and 5 h postprotein changes significantly lower in hypertensives.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Plasma free dihydroxyphenylalanine (DOPA), DOPA sulfate, and free dopamine changes induced 1-5 h after protein ingestion in control (open bars) and hypertensive (hatched bars) subjects. Changes are expressed in percentage ± SE of baseline values (100%) that were within normal limits and without difference between control and hypertensive subjects. Baseline values of plasma free DOPA, DOPA sulfate, and free dopamine were 8.4 ± 0.6, 4.1 ± 0.8, and 0.24 ± 0.05 pmol/ml in control subjects and 11.4 ± 2.8, 5.7 ± 0.7, and 0.34 ± 0.06 pmol/ml, respectively, in hypertensive subjects. * P < 0.05 for intragroup difference from baseline or hour-to-hour changes;  P < 0.05 for difference between controls and hypertensives.

Table 2 summarizes the changes of plasma tyrosine, dopamine sulfate, free and sulfated NE, and 3-O-methyl-DOPA, the latter determined only in controls. With all baseline values normal and comparable in both groups there was a progressive increase in tyrosine and dopamine sulfate after protein intake comparable in both groups but no change in the control period. The plasma dopamine sulfate-to-free dopamine ratio variation 5 h after protein intake was higher (P < 0.02) in hypertensive (86 ± 29) than control subjects (55 ± 14). Plasma free NE and NE sulfate, as well as 3-O-methyl DOPA (determined only in controls), did not change after protein or in the control period. This contrasted with the Epi changes (Fig. 2). Plasma free Epi increase from baseline values after protein in control subjects was significantly different from no change or tendency to decrease observed in hypertensive subjects; on the other hand, there was significantly more Epi sulfate increase in hypertensives than in controls. A similar dichotomy was seen, with a slight delay, mostly after hour 2, in the urinary excretions of free and sulfated Epi. PRA showed an hour-to-hour decrease 2 h after protein in control subjects (without change in the control period) but no change in hypertensive subjects. Plasma aldosterone also decreased in controls 4 h after protein but tended to increase in hypertensives, the difference becoming significant 4 h after protein intake. Plasma ANF was higher in hypertensives at baseline in both settings; 2 h after protein ANF decreased in hypertensives in significant contrast to changes in control subjects. No such changes or differences were observed in the control period.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Plasma and urinary free and sulfated epinephrine (Epi) changes induced 1-5 h after protein ingestion in control (open bars) and hypertensive (hatched bars) subjects. Changes are expressed in percentage ± SE of baseline values (100%) that were within normal limits and without difference between control and hypertensive subjects. Statistical symbols the same as on Fig. 1. Baseline values of plasma free Epi and Epi sulfate were 0.16 ± 0.02 and 0.57 ± 0.07 pmol/ml in controls and 0.27 ± 0.02 and 0.57 ± 0.03, respectively, in hypertensives. Baseline values of urinary free and sulfated Epi were 4.85 ± 0.5 and 6.5 ± 1.2 nmol/h in controls and 4.7 ± 0.4 and 7.2 ± 1.4 nmol/h, respectively, in hypertensives. * P < 0.05 for intragroup difference from baseline or hour to hour changes;  P < 0.05 for difference between controls and hypertensives.

Urinary changes (Fig. 3) from normal and comparable baseline values (free DOPA and dopamine) in both groups showed a significant increase in urinary free DOPA excretion 2 h postprotein followed by a decrease 5 h postprotein in the control group without change in hypertensive subjects. This was associated with a moderate but not significant increase in urinary free dopamine in controls without any change in hypertensive subjects. The urinary free dopamine increase after protein became 4 h postprotein higher in controls than hypertensive subjects only with borderline significance (P < 0.0559). Urinary Na⁺ excretion, having comparable baseline excretions 24 h before protein load (146 ± 16 mmol/24 h in controls, 168 ± 17 in hypertensive subjects), increased after protein intake in hypertensives less than in controls, significantly so 3 h after protein, followed by a decrease in both groups 4 h after protein. The creatinine clearance (baseline 113 ± 4.8 in controls vs. 109 ± 4.8 ml · min¹ · m² in hypertensive subjects), which tended 1 h after protein to decrease in both groups, increased more in controls than hypertensives, significantly so 3 and 4 h after protein intake. No similar inter- or intragroup changes were seen in the control periods.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Urinary free DOPA, dopamine, urinary sodium, and creatinine clearance in control (open bars) and hypertensive (hatched bars) subjects. Changes are expressed in percentage ± SE of baseline values (100%) that were within normal limits and without difference between control and hypertensive subjects. Baseline values of urinary free DOPA and free dopamine excretion were 13.2 ± 2.1 and 76.4 ± 5.8 nmol/h in controls and 14.7 ± 4 and 83.6 ± 6.4 nmol/h, respectively, in hypertensives. * P < 0.05 for intragroup difference from baseline or hour-to-hour changes;  P < 0.05 for difference between controls and hypertensives.

Other urinary changes from normal and comparable baseline values (Table 3) consisted in urinary DOPA sulfate, 3-O-methyl-DOPA, and significant dopamine sulfate increases after protein intake compared with the baseline or control period but without intergroup difference. There were neither intergroup differences nor protein-induced changes in the urinary excretion of HVA and DOPAC. Urinary free NE excretion changes after protein intake or equivalent hydration tended to be higher (not significantly) in controls than in hypertensives, probably due to their higher creatinine clearance increase. Urinary NE sulfate excretion increased in response to protein intake in controls but not hypertensives, a difference significant 1 h after protein. The urinary dopamine-to-DOPA ratio increased 1 h after protein intake in controls, but not in hypertensives, and exhibited a high variability in the control period.

The correlation coefficients calculated between all variables showed the following.

First, in control subjects there is a positive correlation between urinary Na⁺ excretion and plasma free dopamine baseline and their change 1 h after protein (r = 0.65, P < 0.009; r = 0.59, P < 0.02) as well as a correlation between urinary Na⁺ excretion and urinary dopamine sulfate excretion baseline (r = 0.49, P < 0.039); there are no such correlations in hypertensive subjects (r = 0.08, 0.07, 0.18, respectively). It is noteworthy that no correlation was found between urinary Na⁺ and free dopamine excretion in baselines of control and hypertensive subjects and any control period before or after protein intake.

Second, baseline plasma free dopamine was 1) positively correlated with plasma free Epi in control (r = 0.86, P <0.0001) but not in hypertensive subjects and 2) negatively correlated with plasma DOPA sulfate (r = 0.97, P < 0.02) and dopamine sulfate (r = 0.68, P < 0.005) in control but not in hypertensive subjects.

Third, baseline plasma free Epi was negatively correlated with plasma DOPA sulfate in controls (r = 0.86, P < 0.03) but not in hypertensives.

Fourth, baseline plasma dopamine sulfate correlated with urinary dopamine sulfate in control (r = 0.86, P < 0.0001) but not hypertensive subjects.

	DISCUSSION

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The increased plasma tyrosine concentration in response to the protein load ingestion was followed in control subjects by an increase of plasma free DOPA, free dopamine, and dopamine sulfates and an increase of the urinary excretion of free DOPA and dopamine sulfate. However, urinary free dopamine increase remained only at the limit of significance. Other metabolites of DOPA (DOPA sulfate and 3-O-methyl-DOPA) and of dopamine (DOPAC and HVA) and plasma and urinary NE remained unaffected, as previously observed (27). Protein intake-induced increased plasma dopamine sulfate was associated to a lesser degree with an Epi sulfate increase. These tyrosine and plasma free DOPA and dopamine sulfate data are in accordance with previous observations (8, 9) that a substantial amount of DOPA and dopamine sulfate, but not free NE, is derived from meals. The concept of an apparently nutritional priming-induced free dopamine increase is also supported by a recent study (20) in rats in which a protein feeding-induced free dopamine increase was demonstrated to be solely catecholamine precursor dependent.

The main new observation in this study is the plasma free dopamine increase and its correlation with early natriuresis after protein intake (as well as urinary dopamine sulfate excretion increase paralleling natriuresis), whereas there is only a borderline increase in urinary free dopamine unrelated to sodium excretion. These urinary free dopamine and sodium results different from a previous study (27) may be due to the higher age of controls (close to 50 yr in the present study, about twice that of previously studied), because protein-induced urinary free dopamine excretion and natriuresis decrease with age (9). The less conspicuous urinary free dopamine increase in response to protein intake is in accordance with the lack of evidence that renal dopamine synthesis would mediate renal changes in response to protein in healthy volunteers (23). Another study even described in healthy subjects a decrease in urinary free dopamine after an acute protein intake (15). The plasma free dopamine increase and its relationship to sodium excretion after protein ingestion rather complements previous studies (8, 9, 27), because plasma free dopamine was not determined (9, 27) or mostly not detectable (8) in these studies. They used HPLC techniques, which are less sensitive than the radioenzymatic method (25), resulting in plasma free dopamine usually decreasing below detection limit. The plasma free dopamine increase after protein ingestion suggests that, despite massive sulfoconjugation of dopamine in autocrine-paracrine sources (11) (probably the gut), a small portion of free dopamine generated after protein ingestion may escape into the circulation. Even if the picomolar range of free dopamine increase may not be sufficient to arouse dopamine type 1 receptors, the renin decrease 2 h postprotein followed by aldosterone decrease 4 h postprotein do not exclude an indirect natriuretic effect due to an inhibitory action of dopamine on renin (28) and aldosterone (3). The absence of ANF increase after protein confirms that ANF is not involved in the hyperfiltration after a high- protein meal (21).

The protein-induced parallel increases of free dopamine and Epi (unlike free NE) are reminiscent of a similar parallelism in urinary catecholamine increases in protein-fed rats (14). In the widely distributed autocrine-paracrine system with the mRNA to tyrosine hydroxylase expressed (11), the protein-generated tyrosine may be converted by tyrosine hydroxylase to DOPA. The better circulatory accessibility of both precursors to autocrine-paracrine and adrenomedullary than less blood-supplied sympathetic nerve terminal structures probably contributes to this differential response. A similar concomitant increase of dopamine and Epi and their sulfates, respectively, was also observed in tyrosine and L-DOPA-loaded healthy subjects (5). Dopamine and Epi also have in common a "hormonal" secretory character distinct from NE (a recognized poor postprandial marker; Ref. 24) and higher affinity of both toward phenolsulfotransferase than that of NE (1). This makes dopamine and Epi more susceptible to sulfoconjugation occurring predominantly in blood (2) than the mostly intraneuronally inactivated NE.

The most apparent difference of hypertensive patients from controls is that despite having comparable plasma tyrosine and dopamine sulfate increases, they did not increase plasma and urinary free DOPA and dopamine. Instead, their DOPA sulfate increase became predominant without any difference in other DOPA and dopamine metabolites; only the plasma dopamine sulfate increase was disproportionally high when related to plasma free dopamine as reflected by a higher late postprandial plasma dopamine sulfate-to-free dopamine ratio. These abnormalities were coupled with an attenuated urinary sodium and creatinine clearance increase compared with controls and a loss of the positive correlation between plasma free dopamine and urinary sodium in hypertension. An earlier (1-2 h postprandial) shift from free to sulfoconjugated Epi, the latter increasing more in hypertensives at the expense of free Epi (but no changes in plasma free and conjugated NE), was also observed.

The failure to increase free DOPA after protein ingestion in hypertension appears to be in the present setting most compatible with excess free DOPA becoming sequestrated in hypertension in the form of DOPA sulfate (6). The causes of the DOPA sulfate increase, partially paralleling dopamine and Epi sulfate increases in hypertension, remain unexplained. DOPA sulfate increase may be due to a decreased renal clearance of DOPA sulfate similar to a decreased renal clearance of dopamine sulfates in hypertension in control subjects (12); this may explain the loss of the observed positive correlation between plasma and urinary dopamine sulfate in hypertension. Plasma levels of DOPA sulfate appear to be particularly dependent on their renal clearance, because their estimated fractional renal excretion is approximately 10-fold higher than that of dopamine sulfate (17). A more general decreased renal clearance of catecholamine conjugates in hypertension may also account for a lower urinary NE sulfate increase after protein intake in hypertensives than in controls. Whatever the mechanism of the higher sulfoconjugation propensity in hypertension, it probably results in less free DOPA becoming postprandially available for free dopamine generation in autocrine-paracrine tissues and to be taken up by the adrenal medulla for free Epi generation. This may contribute, with an early postprandial shift from free Epi to Epi sulfate followed by a later shift from free dopamine to dopamine sulfate, to lower postprotein plasma free Epi and dopamine concentrations in hypertension.

The clear failure to increase plasma free dopamine after protein intake was associated in hypertension with a defective postprandial natriuresis and diuresis but urinary free dopamine excretion differences only at the limits of significance compared with control subjects. This urinary free dopamine finding different from a previous study (4) may be due to only six hypertensive subjects being studied (compared with 18 in this study), with hypertensive subjects having twice as high urinary baseline natriuresis as controls (unlike the present comparable baseline). Minor renal dopamine generation abnormalities may nevertheless exist in hypertensives as suggested by their failure to have an early increase of the urinary dopamine-to-DOPA ratio (indicating less dopamine generation from DOPA) in response to protein. A hyporesponsiveness of this ratio to salt was also observed in salt-sensitive hypertension (10). An additional contributor to lower postprotein sodium excretion in hypertensives may be their failure to suppress aldosterone unlike the controls. This may be due to their inability to stimulate by protein intake plasma free dopamine, an inhibitor of the aldosterone response to angiotensin II (3). The aldosterone increase 4 h after protein in hypertensives without corresponding renin increase may thus result from a dopamine defect-related aldosterone disinhibition from the stimulatory renin-angiotensin control. Baseline plasma ANF concentrations were higher as previously observed in hypertension (18) with the only difference that in hypertensives the ANF response to protein intake was inferior to that in controls. This may be an additional contributor to the decreased natriuresis. The attenuated natriuresis, diuresis, and creatinine clearance increase after protein ingestion in the presence of a comparable oral hydration resulted in hypertensive subjects in a moderate hemodilution. This was reflected by plasma albumin decreasing to values lower than in controls without comparable changes in the control periods without protein but the same oral hydration.

The systolic blood pressure decrease in hypertensives and pulse rate increase in controls is due to protein intake, because none of these changes occurred in the control period without protein. The lower protein-induced free Epi increase in hypertensives is compatible with an attenuated -adrenergic (pulse rate-reflected) response compensatory to the postprandial blood pressure decrease. It is not clear to what degree this attenuated Epi response in hypertension is specific for protein intake. The postprandial heart rate response to oral glucose proved also to be attenuated in hypertension, but only free NE had been determined and found to be hyporesponsive in this study (29).

Perspectives

In hypertension, the association of the failing postprandial natriuretic and pulse rate responses with a failure to increase plasma free DOPA, dopamine, and Epi coupled with a yet unexplained excess of DOPA, dopamine, and Epi sulfates raises the possibility of a causal relationship between excessive sulfoconjugation and lower availability of the respective free catecholamine for biological action. Because tyrosine increase after protein is normal in hypertension, the next step, DOPA and the balance of its free and conjugated fraction will merit particular attention, because the failure to postprandially increase plasma free DOPA in hypertensive subjects appears to be the culprit of their failure to increase plasma free dopamine and free Epi in response to protein ingestion.