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Original Studies |
Research Institute of Child Nutrition, Dortmund (T.R., F.M.), Institute of Nutrition, Department of Pathophysiology of Nutrition, University of Bonn (K.P.), Bonn, Germany
Address all correspondence and requests for reprints to: Dr. Thomas Remer, Forschungsinstitut für Kinderernährung, Heinstück 11, 44225 Dortmund, Germany.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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-androstanediol glucuronide with diet P. Urinary 24-h
excretion rates for C peptide, free cortisol, DHEAS, and total
17-ketosteroid sulfates were clearly reduced with diet L compared to
those with diet N or P. Our results show that a lactovegetarian diet
can reduce adrenocortical activity (at least after a short term diet
change). In addition, this vegetarian nutrition leads to a particular
metabolic situation (elevated plasma DHEAS and reduced urinary DHEAS
output) that usually is characteristic of fasting. Peripheral androgen
metabolism as reflected by urinary 3-androstanediol glucuronide
appears to be influenced only by high protein intake (diet P). Further
research (controlled dietary long term investigation) is required 1) to
validate whether the effects of diet on adrenocortical activity
represent sustained endocrine changes and 2) to elucidate the
underlying mechanism. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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To address this issue, the hormonal responses to different, nearly isoenergetic diets [normal (N), protein rich (P), and lactovegetarian (L)] were investigated in a controlled diet study. Dietary periods of 5-day duration were chosen to clearly identify short term (not acute) effects and to assure full dietary compliance. Recent nutritional as well as endocrinological studies have shown that plasma levels and 24-h excretion rates of AA can change within periods of a few days after distinct manipulations (11, 12). In contrast, acute responses of serum DHEAS to dietary manipulation can even occur after 1–2 h, as has been demonstrated, for example, by means of an oral glucose load (5).
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Six healthy adult volunteers (three women, aged 24–25 yr, and three men, aged 31–49 yr) were recruited from the staff of the Research Institute of Child Nutrition. Mean values (±SD) for weight and height were 65.7 ± 5.9 kg and 171.2 ± 8.3 cm, respectively, for women, and 81.1 ± 4.9 kg and 182.6 ± 4.8 cm for males. All subjects had a normal physical examination as well as normal findings from multiparameter urine test strips (Combur8-Test, Boehringer Mannheim, Mannheim, Germany; identical with Chemstrip 8 urine test strips, Boehringer Mannheim, Indianapolis, IN) measuring pH, nitrite, protein, glucose, ketones, urobilinogen, bilirubin, and blood. None had a past medical history of renal, endocrine, or cardiovascular disease. Except for oral contraceptives (OC) containing a constant dose of 35 µg ethinyl estradiol (all of the women), no medications were taken by the subjects during the study. Women using OCs were selected to surely exclude menstrual phase-related fluctuations in endogenous hormones as potential confounders of the diet effects under investigation. The diet periods were timed to maintain a constant exogenous estrogen supply. Using that kind of study protocol we had to accept that the exogenous estrogen intake, which is known to affect endogenous sex hormone-binding globulin (SHBG) and cortisol levels, could obscure some of the diet effects on endocrine parameters. On the other hand, basic endocrine functions, e.g. free cortisol levels and cortisol activity (13) or MCR of DHEA (14), appear to be normal in OC users. The study protocol was approved by the institutional review board of the Dortmund Research Institute of Child Nutrition. Participants gave written informed consent after the experimental protocol was explained to them in detail.
Experimental protocol and diets
Apart from studying AA metabolism, another aim of this study was to investigate whether it is possible to reliably estimate the renal net acid excretion produced by different natural food diets. As the corresponding results on renal net acid excretion have already been published along with a detailed description of study design and diets (15), the latter is briefly summarized here.
The investigation, a controlled experimental diet study (repeated
measure design), was carried out in four consecutive diet periods
during which all subjects received the respective diets in the same
chronological order. Each diet period lasted 5 days. In the initial
period, a normal (N), moderately protein-rich diet (protein, 95 g/day;
fruits and vegetables, 700 g/day) was fed. This was followed by a
protein-rich (P) diet, a lactovegetarian (L) diet, and a repetition of
the initial diet N. Protein contents with diets P and L were 120 and 49
g/day, respectively (Fig. 1
). The amount
of fruits and vegetables ingested with diet P (L) was 230 (1610) g/day.
Intakes of dietary fiber and carbohydrates were both highest with diet
L, but carbohydrate intake differed only weakly between diets L and N
(Fig. 1). The repeat diet N was slightly modified, in that 20 mmol (3.0
g) L-methionine (Acimethin, Gry-Pharma, Kirchzarten,
Germany) were additionally administered per day to increase
specifically renal net acid load. The last three diet periods (P, L,
and repeat N) were each separated by a 9-day interval without dietary
restrictions. A 2-day interval without strict diet regulations was set
between the initial (N) and the second (P) period.
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2 h after the subjects
had completed their first 24-h-urine collection). Analytical procedures
Commercial solid phase 125I RIAs (coated tube
methodology) were used for the measurements of DHEAS, DHEA, cortisol
(these three kits from Diagnostic Products Corp., Los Angeles, CA),
androstenedione (
4A), and 3-androstanediol
glucuronide (AdiolG; 5-androstan-3,17ß-diol glucuronide; both
kits from Diagnostic Systems Laboratories, Webster, TX). C Peptide was
analyzed by RIA using the polyethylene glycol-accelerated, double
antibody method to separate the bound 125I-labeled C
peptide from the unbound fraction (Diagnostic Products Corp.).
Insulin-like growth factor I (IGF-I) was quantified with an
immunoradiometric 125I assay (Diagnostic Systems
Laboratories), and SHBG was determined with an enzyme immunoassay (SHBG
MTPL EIA, DRG Instruments, Marburg, Germany). Albumin determination was
performed colorimetrically using the bromocresol purple procedure
(albumin bicinchoninic acid, Sigma Chemical Co., St. Louis, MO).
Urinary creatinine output was measured by the Jaffé method using
a Beckman-2 creatinine analyzer (Beckman Instruments, Fullerton,
CA).
The measurements of plasma hormones, plasma albumin, SHBG, and urinary C peptide were carried out according to the respective kit instructions. Urinary DHEAS was quantified directly (like plasma DHEAS), without kit modification or specific sample preparation (16). Quantification of urinary AdiolG was performed as specified previously (12). Before urinary cortisol measurement, the glucocorticoid was extracted with methylene chloride as previously described (12). Urinary total 17-ketosteroid sulfates (17-KSS) were measured without previous hydrolysis (as conjugated Zimmermann chromogens) after C18 reverse phase extraction and LH-20 chromatography (17). Intra- and interassay coefficients of variation for the various immunological steroid measurements were below 11% each, and those for C peptide and IGF-I were below 8% and 17%, respectively. Intra- and interassay precisions for 17-KSS did not exceed 15% and 18%, respectively.
Statistical analysis
Two-way ANOVA for repeated measurements was applied to study the
effects of diet on the measurement variables. The independent grouping
factor was sex. Comparisons between the means of paired observations
with different dietary treatments were evaluated by linear contrasts. F
statistic values are provided wherever repeated measures ANOVA-derived
P values were significant (P < 0.05). In
case that sphericity assumption for repeated measures ANOVA was not met
(this was the case for plasma DHEAS, Table 1), a paired t
test for a single comparison between two means was performed instead.
In this case (i.e. for plasma DHEAS), mean values were given
separately for males and females without further test for sex
difference. For all other analytes, separate means for females and
males were presented only if the grouping factor sex (of ANOVA) yielded
P < 0.1. Data are presented as the mean ±
SD.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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). The only
exception was circulating DHEAS, which was significantly elevated with
diet L compared to diet N. No diet effect occurred for IGF-I and SHBG,
but albumin was significantly reduced with diet P (Table 1). Sex
differences were seen in the circulating steroids cortisol and AdiolG,
but not DHEA or androstenedione (Table 1). Also, for the plasma
proteins albumin and SHBG, clear sex differences were discernible.
Urinary 24-h excretion rates for DHEAS (Fig. 2), total 17-KSS (Table 2), cortisol (Fig. 3), C peptide, and creatinine (Table 2)
were clearly reduced with diet L compared to those with diet N. A sex
difference was no longer discernible for renal output of cortisol, but
appeared for urinary DHEAS and was also present for 17-KSS and
creatinine (Table 2). An increase of about 60% was seen for the
urinary 24-h output of AdiolG (Fig. 4)
with diet P in both males and females. This percent increase was almost
comparable to the urinary AdiolG response seen after a 3-day hCG
stimulation test in healthy males (12). Women had a markedly reduced
overall 24-h AdiolG excretion rate compared to that in men (Fig. 4).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The parallel decreases in urinary excretion of DHEAS and 17-KSS, on the
one hand, and cortisol, on the other hand, seen with diet L, strongly
suggest that the underlying mechanism responsible for the urinary AA
reduction with vegetarian nutrition could be a reduced adrenocortical
activity, i.e. a simultaneous decrease in adrenal
glucocorticoid and AA secretion. The additional decrease in urinary
creatinine excretion of about 19%, on the average (Table 2), is
obviously due to the elimination of meat intake (including meat
products) with diet L. Cooked meat contains considerable amounts of
preformed creatinine and creatine, which, after ingestion, lead to
diet-induced acute and long term increases in urinary creatinine
excretion of 10–30% (18, 19). Thus, the direct stimulus of diet
composition, especially meat intake, on urinary creatinine output would
obscure specific endocrine effects if urinary steroid data were related
to urinary creatinine output and not presented as 24-h excretion
rates.
As the cholesterol content of the diets L and N differed only slightly [estimated cholesterol intakes according to the food composition tables of Souci et al. (20), 123 mg/day with diet L and 233 mg/day with diet N], it does not appear plausible that a reduced cholesterol supply could have affected adrenal steroidogenesis. Accordingly, there were no marked differences in the plasma or urinary AA and cortisol patterns between diets N and P, although cholesterol intake was clearly elevated with diet P (580 mg/day). In addition, the plasma cholesterol-lowering effect of a high fiber intake maintained for only a few days is very modest (11). Recently, Arem et al. (21) provided indirect evidence that adrenal steroidogenesis does not respond in a particularly sensitive manner to changes in circulating cholesterol levels. The researchers observed that even in severe acquired low density lipoprotein cholesterol deficiency, glucocorticoid secretion is impaired only slightly.
The drop in urinary cortisol and AA excretion occurring with diet L was not associated with a parallel drop in circulating hormone concentrations. This finding is not inconsistent for the following reasons.
First, urinary 24-h cortisol excretion integrates the plasma free cortisol concentrations during the whole day and is therefore regarded as the most direct and reliable practical index of cortisol secretion (22, 23). Plasma concentrations of cortisol, on the other hand, not only vary in the diurnal course but are also subject to a considerable pulsatility (24).
Second, hepatic secretion of the major binding protein for cortisol, the corticosteroid-binding globulin, frequently differs from adrenocortical activity. For example, estrogens, whether originating from oral contraceptives or endogenous secretion, increase corticosteroid-binding globulin levels and thus raise total plasma cortisol without raising cortisol activity (13). This is also reflected in comparable urinary cortisol excretion rates but markedly differing cortisol plasma levels between the male and female volunteers and it implies again that changes in adrenocortical activity are not inevitably detectable by plasma cortisol measurements.
Third, a slowed steroid metabolism or plasma clearance could also mask a reduced hormone secretion, leaving plasma hormone levels almost unchanged. The plasma clearance of steroids can even be reduced so drastically that hormone concentrations significantly increase despite a strong reduction in hormone secretion and excretion. This is especially the case for DHEAS in fasting subjects (1, 2, 3). As is obvious from our study also, the isocaloric switch to vegetarian nutrition (diet L) can lead to the kind of particular metabolic situation with elevated plasma DHEAS levels and reduced urinary DHEAS output that is normally characteristic of fasting.
There is increasing evidence that insulin stimulates the MCR of both
DHEA and DHEAS, resulting in lowered plasma DHEA(S) levels after
insulin increases (5, 10, 25, 26). As discussed previously (27), this
insulin effect on circulating DHEA(S) does not appear to be sex
specific. Also, the elevated plasma DHEAS level with diet L (in the
present study) was associated with a significantly reduced overall
insulin production during the day, as reflected by urinary C peptide
excretion. Whether this reduction in insulin secretion is solely
responsible for the increase in plasma DHEAS with diet L is
questionable, because changes in plasma DHEAS levels are not always
seen when insulin secretion varies (28, 29). In addition, dietetically
induced variations in plasma DHEAS are not necessarily associated with
alterations in insulin secretion and DHEAS excretion, as was recently
observed in an experimental diet study with an isolated rise in dietary
fiber (pectin) intake resulting in increased plasma DHEAS and
unaffected plasma DHEA (11). Thus, the increased dietary fiber intake
with diet L could also have contributed to the elevated plasma DHEAS by
slowing especially the plasma clearance of DHEAS without affecting DHEA
and 4A levels.
Mounting evidence suggests that insulin not only has a stimulatory effect on AA metabolism, but is also involved (along with IGF-I) in the regulation and enhancement of cortisol and AA secretion (30, 31, 32, 33). In contrast to this view several researchers conclude that insulin instead decreases AA secretion. Decreased plasma levels of AA are frequently observed in association with elevated plasma insulin and vice versa (5, 10, 34, 35, 36). However, the respective contributions of DHEAS clearance and DHEAS secretion to the observed plasma DHEAS levels have scarcely been considered together in these studies. If, hypothetically, an insulin-induced rise in AA secretion turns out to be lower than the parallel induced rise in the MCR, plasma levels of AA would fall, and interpreting the latter as a decline in AA secretion would be wrong.
With regard to our finding of a decline in adrenocortical activity with the vegetarian diet, we favor the idea that the drop in insulin secretion could be involved as a causal factor. Urinary C peptide excretion did only show a moderate reduction, but in contrast to the recently seen unresponsiveness of urinary and circulating DHEAS to a similar C peptide decrease (29), an additional nutritional factor known to affect the insulin secretory dynamics was effective in the present study. This factor was dietary fiber intake that along with protein intake showed the strongest percent difference between diet L and diet N. A decrease in protein intake per se reduces insulin secretion (29, 37), and an increased fiber (especially pectin) intake further attenuates postmeal insulin secretory peaks (38, 39). Integrated insulin secretion, i.e. urinary 24-h C peptide output, is not necessarily affected by this insulin peak-flattening effect of fiber, because an altered postprandial plasma dynamics with a slowed glucose and insulin decline can be present (38). Thus, if insulin is actually involved in the modulation of adrenocortical activity as suggested here, the sole variation of only one potent dietary insulin secretagogue [with definitely stronger effects on C peptide excretion, as recently described (29)] should also produce significant changes in urinary cortisol and DHEAS excretion. This hypothesis remains to be checked by future studies.
In contrast to our short term diet experiment, other studies have shown significant SHBG increases with vegetarian nutrition (7, 40) or with a high carbohydrate and low protein diet (9). This discrepancy is eventually due to the fact that in these studies diet periods longer than 5 days were investigated. Regardless of study duration, findings on decreased plasma DHEAS and cortisol (10) or unaffected plasma DHEAS levels (41) with low fat and high carbohydrate cannot be directly compared with our results because we did primarily vary the protein/carbohydrate ratio.
Whether the significant decrease in plasma albumin with diet P,
probably indicating an increased albumin catabolism, is of
physiological relevance for AA metabolism cannot be finally answered,
but it appears possible that this albumin decline entails the delivery
of rising amounts of freed androgen precursor molecules (mainly DHEAS
and DHEA) to androgen target tissues. Indeed, an increase of about 60%
was seen for the 24-h output of AdiolG with diet P. Along with
androsterone glucuronide, AdiolG is one of the major 5-reduced
metabolites of androgen metabolism (26, 42, 43), and its urinary rise
suggests an increased MCR of the adrenal and gonadal steroid precursors
DHEA(S), 4A, and testosterone (42). The unchanged plasma
AdiolG levels with the different diets are in accord with the findings
of Giagulli et al. (42) and Lavallée et al.
(26), who both observed a striking unresponsiveness of plasma AdiolG
despite a definite increase in 5-reductase-related C19
steroid metabolism as verified by either elevated plasma levels of
androsterone glucuronide after an infusion of DHEA and insulin (26) or
elevated 24-h urinary excretion rates of androsterone glucuronide in
moderately obese subjects compared to those in nonobese controls
(42).
Thus, our findings indicate that variations in plasma levels of certain steroids (AdiolG, DHEAS, and cortisol) do not always reflect their changes in bioactivity, blood production, and/or secretion, whereas urinary 24-h excretion rates appear to be more sensitive indexes. However, to definitely decide this issue, direct determinations of secretion and clearance rates would be needed. Another implication of our short term study is that it could offer a possible explanation for the higher longevity frequently seen in vegetarian populations (44), as a continuously reduced adrenocortical activity might contribute to a delay in the onset of various chronic degenerative diseases. In this context it is noteworthy that aging appears to be associated with a disadvantageous progressive elevation of basal cortisol levels and a reduction in the nocturnal quiescent periods of adrenal secretory activity (45).
In summary, switching to a vegetarian diet can lead to a reduction in adrenocortical activity, at least as reflected by urinary excretion rates of adrenal steroid hormones after a short term diet change. The respective hormonal changes appear to be attributable to combined food effects, probably an increased dietary fiber intake and a decreased protein intake. Isolated changes in single dietary components (dietary fiber and protein) did not induce comparable results (11, 29). Peripheral androgen metabolism as reflected by urinary AdiolG was not specifically reduced by the lactovegetarian diet (i.e. the highest dietary fiber/protein ratio), but was clearly stimulated by the highest protein intake. Further investigations, controlled dietary long term intervention studies, as well as determinations of hormonal secretion and clearance rates are required 1) to validate whether the observed diet effects represent sustained changes in adrenocortical activity and 2) to elucidate the eventual role of insulin in the regulation of AA and cortisol secretion.
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Footnotes |
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Received December 8, 1997.
Revised February 19, 1998.
Accepted February 26, 1998.
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References |
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,17ß-diol glucuronide as a steroid correlate of
visceral obesity in men. J Clin Endocrinol Metab. 82:1528–1534.This article has been cited by other articles:
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  T. Remer, T. Dimitriou, and C. Maser-Gluth Renal Net Acid Excretion and Plasma Leptin Are Associated with Potentially Bioactive Free Glucocorticoids in Healthy Lean Women J. Nutr., February 1, 2008; 138(2): 426S - 430S. [Abstract] [Full Text] [PDF]
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A. E. Buyken, Y. Kellerhoff, S. Hahn, A. Kroke, and T. Remer Urinary C-Peptide Excretion in Free-Living Healthy Children Is Related to Dietary Carbohydrate Intake but Not to the Dietary Glycemic Index J. Nutr., July 1, 2006; 136(7): 1828 - 1833. [Abstract] [Full Text] [PDF]
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 M. F McCarty Insulin and IGF-I as determinants of low 'Western' cancer rates in the rural third world Int. J. Epidemiol., August 1, 2004; 33(4): 908 - 910. [Full Text] [PDF]
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 T. Remer CCN Proteins Are Distinct from, and Should Not Be Considered Members of, the Insulin-Like Growth Factor-Binding Protein Superfamily J. Clin. Endocrinol. Metab., February 1, 2001; 86(2): 950 - 950. [Full Text]
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T. Remer and F. Manz Role of Nutritional Status in the Regulation of Adrenarche J. Clin. Endocrinol. Metab., November 1, 1999; 84(11): 3936 - 3944. [Abstract] [Full Text]
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