• View in gallery
    Figure 1—

    Mean ± SEM serum concentrations of estradiol after ACTH administration in dogs that were evaluated before (0 months) and after (12 months) receiving an HID (n = 10) or LID (10) for 12 months. *Values at indicated time are significantly (P < 0.001) different.

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Effects of dietary soy isoflavones on health, steroidogenesis, and thyroid gland function in dogs

Rosario CerundoloDepartment of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104.

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Kathy E. MichelDepartment of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104.

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Michael H. CourtDepartment of Pharmacology and Experimental Therapeutics, School of Medicine, Tufts University, Boston, MA 02111.

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Binu ShresthaDepartment of Pharmacology and Experimental Therapeutics, School of Medicine, Tufts University, Boston, MA 02111.

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Kent R. RefsalDiagnostic Center for Population and Animal Health, College of Veterinary Medicine, Michigan State University, Lansing, MI 48824.

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Jack W. OliverClinical Endocrinology Service, Department of Comparative Medicine, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37966.

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Vincent BiourgeRoyal Canin Research Center, BP 4, 30470, Aimargues, France.

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Frances S. ShoferDepartment of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104.

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Abstract

Objective—To evaluate the effect of a soy-based diet on general health and adrenocortical and thyroid gland function in dogs.

Animals—20 healthy privately owned adult dogs.

Procedures—In a randomized controlled clinical trial, dogs were fed a soy-based diet with high (HID; n = 10) or low (LID; 10) isoflavones content. General health of dogs, clinicopathologic variables, and serum concentrations of adrenal gland and thyroid gland hormones were assessed before treatment was initiated and up to 1 year later. Differences between groups with respect to changes in the values of variables after treatment were assessed by means of a Student t test (2 time points) and repeated-measures ANOVA (3 time points).

Results—No differences were detected between the 2 groups with respect to body condition and results of hematologic, serum biochemical, and urine analyses. Most serum concentrations of hormones did not change significantly after treatment, nor were they affected by diet. However, the mean change in serum concentration of total thyroxine was higher in the HID group (15.7 pmol/L) than that in the LID group (–1.9 pmol/L). The mean change in estradiol concentration after ACTH stimulation at 1 year after diets began was also higher in the HID group (19.0 pg/mL) than that in the LID group (–5.6 pg/mL).

Conclusions and Clinical Relevance—Phytoestrogens may influence endocrine function in dogs. Feeding soy to dogs on a long-term basis may influence results of studies in which endocrine function is evaluated, although larger studies are needed to confirm this supposition.

Abstract

Objective—To evaluate the effect of a soy-based diet on general health and adrenocortical and thyroid gland function in dogs.

Animals—20 healthy privately owned adult dogs.

Procedures—In a randomized controlled clinical trial, dogs were fed a soy-based diet with high (HID; n = 10) or low (LID; 10) isoflavones content. General health of dogs, clinicopathologic variables, and serum concentrations of adrenal gland and thyroid gland hormones were assessed before treatment was initiated and up to 1 year later. Differences between groups with respect to changes in the values of variables after treatment were assessed by means of a Student t test (2 time points) and repeated-measures ANOVA (3 time points).

Results—No differences were detected between the 2 groups with respect to body condition and results of hematologic, serum biochemical, and urine analyses. Most serum concentrations of hormones did not change significantly after treatment, nor were they affected by diet. However, the mean change in serum concentration of total thyroxine was higher in the HID group (15.7 pmol/L) than that in the LID group (–1.9 pmol/L). The mean change in estradiol concentration after ACTH stimulation at 1 year after diets began was also higher in the HID group (19.0 pg/mL) than that in the LID group (–5.6 pg/mL).

Conclusions and Clinical Relevance—Phytoestrogens may influence endocrine function in dogs. Feeding soy to dogs on a long-term basis may influence results of studies in which endocrine function is evaluated, although larger studies are needed to confirm this supposition.

Phytoestrogens are plant-derived, nonsteroidal compounds that possess estrogenic activity. There are 3 classes of phytoestrogens, one of which, the flavonoids, contains isoflavones. The principal dietary source of isoflavones is soybeans, which contain the compounds in 4 related chemical structures: the aglycones, the 7-O-glucosides, the 6-O-acetylglucosides, and the 6'-O-malonylglucosides.1

Soy is a common ingredient in commercial dog foods, and many of these foods contain phytoestrogens.2 It has been speculated that consumption of phytoestrogen-rich diets could influence adrenal or gonadal steroidogenesis and thyroid gland function. Phytoestrogens may impair fertility and predispose humans and some other animals to reproductive tract disorders.3 Rats fed a soy diet have delayed onset of sexual maturity.4 Soy isoflavones such as daidzein and genistein accumulate in target tissues (eg, liver or adrenal glands) and inhibit activities of 3B–hydroxysteroid dehydrogenase, aromatase, and 17B–hydroxysteroid dehydrogenase, all of which are enzymes involved in sex hormone steroidogenesis.5,6 Short-term administration of dietary soy reportedly has a measurable, although modest, effect on homeostasis of thyroid gland hormones in cats.7

Investigation of the effect of soy isoflavones on health and hormonal status in dogs has been limited. The purpose of the study reported here was to evaluate the effect of a soy-based diet on general health and adrenocortical and thyroid gland function in adult dogs. Specifically, we hypothesized that consumption of foods containing soy isoflavones would influence the hormonal status of dogs, thereby inducing alteration of steroidogenesis and production of thyroid gland hormones.

Materials and Methods

Animals—Healthy dogs were recruited from among staff and students of the University of Pennsylvania veterinary teaching hospital. Pet owners were informed about procedures, and written consent was obtained. Eligible dogs were required to be healthy as judged on the basis of medical history, physical and dermatologic examinations, and clinicopathologic tests. The physical examination and clinicopathologic analyses were also repeated at the end of the study. Dogs were also required to be between 2 and 8 years of age and neutered. Dogs with a curly coat type (eg, poodles) or with systemic or endocrine or dermatologic diseases were excluded. The study protocol was reviewed and approved by the hospital protocol review and the institutional animal care and use committees.

Experimental design—A sample size of 10 dogs/group was selected so that the study would be able to detect a 40% difference in serum estradiol concentration with 80% power (A = 0.05). However, 30 dogs were recruited in anticipation of a 30% attrition rate. After recruitment, dogs were assigned to 1 of 2 diets by means of a table of randomly generated numbers. Dogs were fed a commercially available hydrolyzed soy isolate–based dieta (HID) or the same diet with an isolate in which isoflavones had been extracted (LID; Appendix). Preparation of the low-isoflavone soy isolate involved alcohol extraction rather than typical water extraction. Amount fed to each dog was calculated according to a formula (132 kcal metabolizable energy/kg0.75 of body weight) and was adjusted to maintain body weight. The dry, expanded diets were manufactured by 3 companies and were identified by a 3-letter code, thereby blinding all investigators to the isoflavone content of each. Two batches of food were manufactured and supplied in 4 shipments during the year-long study. The shelf life of each diet was at least 12 months.

Owners of participating dogs were instructed to use only soy-free treats, and a list of approved treats was provided to the participants. Owners also completed a questionnaire to evaluate dietary management practices prior to the start of the study, and every month thereafter, they were required to return a form in which they recorded daily, when necessary, any change in their dog's appetite or fecal quality and any additional food given other than the assigned diet and the allowed treats. Owners were asked to pick up more food from investigators at monthly to bimonthly intervals and to discuss any difficulties complying with the diet as problems arose. Palatability was assessed with a 3-point scale (1 = dog refuses the food; 2 = dog eats the food as it typically would; and 3 = dog empties the bowl immediately after serving).

Physical and dermatologic examinations, which included measurement of body weight and body condition score (9-point scale, with 1 representing emaciated and 9 representing obese) were performed at the beginning of the study (0 months) and at 6 and 12 months after treatment began.

Sample collection and general laboratory tests— Food was withheld from dogs beginning at 8:00 PM the night before specimens were obtained, and specimens were collected during the subsequent morning (8:00 AM to 12:00 PM). For each dog, blood samples for various purposes were collected via jugular venipuncture at 0, 6, and 12 months. Serum was harvested, and aliquots of serum were stored at −20°C for hormonal assays at separate laboratories. For routine hematologic and serum biochemical analyses, blood samples obtained at 0 and 12 months after treatment began were evaluated. In addition, a blood coagulation profile and ELISAs for dirofilariasis, ehrlichiosis, and Lyme disease were conducted.

Fecal samples were obtained at 0 and 12 months for parasitologic examination. A sample of freshly voided urine was collected by owners or the investigators at 0 and 12 months for standard urinalysis and measurement of concentrations of isoflavones.

Thyroid gland hormone measurements—Evaluations of serum concentrations of T4, T3, fT4, free T3, TSH, and autoantibodies against T4, T3, and thyroglobulin at 0 and 12 months were performed at Michigan State University. Validation data for measurement of serum concentrations of T4 and TSH,8 T3,9 free T3,10 fT4,11 and autoantibodies against T4 and T312 and thyroglobulin13 have been reported.

Adrenal gland hormone measurements—For ACTH stimulation testing, serum harvested from blood samples obtained at 0, 6, and 12 months before and 1 hour after IV administration of 5 μg of tetracosactrin acetateb/kg was evaluated at the University of Tennessee for concentrations of cortisol, 17-hydroxyprogesterone, androstenedione, estradiol, progesterone, testosterone, and aldosterone. The hormone assays used for ACTH stimulation testing consisted of human kits that have been validated for use in dogs.14,15 The analytic sensitivity of the hormone assay is as follows: testosterone, 0.4 ng/mL; androstenedione, 0.05 ng/mL; 17-hydroxyprogesterone, 0.08 ng/mL; progesterone, 0.02 ng/mL; estradiol, 7.2 pg/mL; and cortisol, 0.02 μg/dL.

Isoflavone measurements—Measurement of concentrations of isoflavones in serum at 0, 6, and 12 months and in urine at 0 and 12 months was performed at Tufts University. Serum and urine samples were assayed for concentrations of soy isoflavone (genistein, daidzein, and glycitein) in duplicate. Briefly, 500 ML of urine or serum was mixed with 1.25 μg of biochanin A (internal standard) and 500 ML of 1M phosphate buffer (pH, 5) and enzymatically deconjugated with 25 ML of β-glucuronidase-sulfatasec at 37°C overnight. Samples were then neutralized with 500 ML of 1nM NaOH,c extracted into 5 mL of ethyl acetate, and dried. After reconstitution in 200 ML of mobile phase solution, samples were analyzed via high-performance liquid chromatography with mass spectrometric detection and an atmospheric pressure chemical ionization source.d Initial mobile phase conditions consisted of 0.5% formic acid in water with 30% methanol at a flow rate of 300 ML/min, with a linear gradient to 100% methanol over 9 minutes. Separation was achieved with a 150 × 2.0-mm 4-Mm column.e Analytes were quantified via positive ion selective reaction monitoring with parent-to-product ion mass (m/z+) transitions of 271 to 215 for genistein, 255 to 199 for daidzein, and 285 to 270 for biochanin A. For quantitation purposes, standard curves were generated by means of pure standards dissolved in a blank matrix. Regression coefficients for standard curves were consistently > 0.98, whereas coefficients of variation of assay duplicates averaged < 20%. The lower limit of quantitation of the assay was 50 nmol/L. Urine isoflavones concentration was expressed relative to urine creatinine concentration (nmol/mg of creatinine) to account for differences in urinary output.

Representative samples of the diets administered in the study were collected at 0 and 6 months for determination of soy isoflavones concentrations (aglycone) by means of a validated method involving acid-methanol hydrolysis followed by high-performance liquid chromatography with UV absorbance detection.16 The limits of quantification of the assay were 1 mg of genistein or daidzein/kg of dry weight of food and 2 mg of glycitein/kg of dry weight of food.

Statistical analysis—To examine differences between diets with regard to changes after treatment with respect to results of hematologic, serum biochemical, and urine analyses as well as hormone concentration analyses, a Student t test was used. In situations in which values differed significantly between diet groups at 0 months, ANOVA was used to adjust for the value at 0 months. For biological variables assessed at 3 points (0, 6, and 12 months), repeated-measures ANOVA was used to compare changes after 0 months between the 2 groups. Data are reported as mean ± SEM unless otherwise indicated. All analyses were performed by use of commercial statistical software.f A value of P < 0.05 was considered significant.

Results

Animals—At the beginning of the study, 14 dogs were allocated to the HID group, and 16 dogs were allocated to the LID group. Ten dogs (4 from the HID group and 6 from the LID group) were subsequently withdrawn from the study for the following reasons: owners of 4 (2 HID and 2 LID) dogs failed to comply with study requirements, 3 LID dogs would not eat the assigned food, 1 HID dog developed a mast cell tumor, 1 LID dog developed retinal degeneration and behavior problems, and 1 HID dog developed aggressive behavior. Twenty dogs completed the study (HID group: 2 females and 8 males; LID group: 4 females and 6 males). There was no significant (P = 0.24) difference in age between HID dogs (mean ± SEM age, 4.1 ± 1.7 years) and LID dogs (5.3 ± 2.6 years).

General physical and dermatologic examinations revealed that all dogs were healthy at the beginning and end of the study. There was no significant change in body condition score between the beginning and end of the study, nor was there a significant (P = 0.70) change in body weight for dogs in the HID group (1.3 ± 0.92 kg) versus those in the LID group (0.8 ± 0.92 kg). Mean food intake during the 12 months was 20.58 ± 2.40 g/kg0.75 of body weight in the HID group and 24.81 ± 1.96 g/kg0.75 of body weight in the LID group.

Routine laboratory tests—At the beginning of the study, routine hematologic, serum biochemical, and urine analyses yielded values within respective reference ranges for most dogs. For all dogs, results of blood coagulation tests were also within respective reference ranges, and results of parasitologic examination of feces were unremarkable. All dogs had negative test results for antibodies against Dirofilaria immitis and Ehrlichia canis, but 7 dogs had positive test results for antibodies against Borrelia burgdorferi. At 12 months after diets were initiated, 6 dogs (4 from the HID group and 2 from the LID group) had slight increases in serum activities of ALT (92 to 262 U/L; reference range, 16 to 91 U/L) and ALP (184 to 2,285 U/L; reference range, 20 to 155 U/L). However, values of these variables did not differ significantly between the 2 time points for each group of dogs, nor did they differ between the 2 groups.

Thyroid gland hormone measurements—With respect to results of hormonal assays, all dogs had values within the respective laboratory reference ranges at the beginning of the study (Table 1). Serum concentrations of fT4 at 0 months differed significantly (P = 0.02) between dogs in the HID and LID groups, but did not differ at 12 months. At 12 months, the only remarkable finding was a serum total T4 concentration that was higher than the upper reference limit in 1 dog from the HID group. Serum concentrations of total T4 did not differ significantly between the HID and LID groups at 0 months (P = 0.06) or at 12 months (P = 0.07).

Table 1—

Results of thyroid gland hormone assays of serum obtained from dogs that were evaluated before (0 months) and after (12 months) receiving an HID (n = 10) or LID (10) for 12 months.

AnalyteDietReference range0 months12 monthsDifferenceP value*
Total T4 (pmol/L)HID15–6726.742.415.70.040
LID33.831.9−1.9
Total T3 (pmol/L)HID1.0–2.51.31.70.40.800
LID1.31.60.3
Free T4 (pmol/L)HID8–2613.214.81.60.005
LID19.915.6−1.3
Free T3 (pmol/L)HID4.5–12.55.75.4−1.30.200
LID5.15.70.6
TSH (ng/mL)HID< 0.680.130.200.070.100
LID0.080.270.18
Antibody against T4 (%)HID< 203.32.9−1.40.500
LID1.02.7−1.2
Antibody against T3 (%)HID< 101.42.00.60.700
LID1.01.10.1
Antibody against thyroglobulin (%)HID< 2017.921.63.70.900
LID13.417.84.4

Difference between groups with respect to change in values from 0 to 12 months was considered significant at P < 0.05.

Results of t tests indicated that changes in serum concentrations of fT4 and total T4 during the study period differed significantly between diet groups (t test; P = 0.005 and 0.04, respectively). However, after statistical adjustment for the significant difference in serum fT4 concentration between groups at 0 months, the difference was no longer evident. Furthermore, after statistical adjustment for the nonsignificant difference in serum total T4 concentration between groups at 0 months, the difference detected via the t test persisted but was no longer significant (P = 0.07).

Adrenal gland hormone measurements—At the beginning of the study (0 months), all dogs had values of serum sex hormone concentrations that were within respective laboratory reference ranges (Table 2). At 12 months, the only remarkable values detected were estradiol concentrations that were slightly higher than the reference range at 0 months (mean ± SD value before ACTH stimulation, 87.10 ± 9.38; mean ± SD value after ACTH stimulation, 87.77 ± 13.08) in 7 dogs (5 from the HID group and 2 from the LID group). Change in serum estradiol concentration from 0 to 12 months was the only variable that differed significantly (P = 0.01) between groups (Figure 1). Dogs fed the HID had an increase in serum estradiol concentration from 0 to 12 months (19.0 pg/mL), whereas dogs fed the LID had a decrease during the study period (–5.6 pg/mL), and this difference between groups was significant (P = 0.001). Statistical adjustment for differences between groups that were detected at 0 months did not affect this result.

Table 2—

Results of assays of adrenal gland hormone concentrations in serum obtained from dogs that were evaluated before (0 months) and after (6 and 12 months) initiation of an HID (n = 10) or LID (10) for 12 months.

AnalyteDietReference rangeEvaluation time
Castrated malesSpayed females0 months6 months12 months
Before ACTH stimulation
   Cortisol (ng/ml)HID2.0–56.52.1–58.828.326.232.2
LID19.432.033.8
   Androstenedione (ng/mL)HID0.1–3.60.1–5.71.72.42.3
LID1.43.13.3
   Progesterone (ng/mL)HID0.01–0.170.01–0.490.270.200.28
LID0.220.210.31
   17-hydroxyprogesterone (ng/mL)HID0.01–0.220.01–0.070.210.350.25
LID0.250.380.32
   Testosterone (ng/mL)HID0.01–0.240.01–0.320.030.020.03
LID0.040.020.03
   Estradiol (pg/mL)HID23.1–65.130.8–69.954.060.066.6
LID45.054.743.1
   Aldosterone (pg/mL)HID3.5–139.93.5–139.9ND35.725.9
LIDND35.044.2
After ACTH stimulation
   Cortisol (ng/mL)HID70.6–151.265.0–174.6116.7119.9122.0
LID111.8109.3110.5
   Androstenedione (ng/mL)HID2.4–29.02.7–39.710.015.214.1
LID10.414.515.4
   Progesterone (ng/mL)HID0.22–1.450.10–1.502.032.061.79
LID1.991.631.61
   17-hydroxyprogesterone (ng/mL)HID0.25–2.630.40–1.621.802.542.08
LID2.012.771.81
   Testosterone (ng/mL)HID0.02–0.420.02–0.450.040.030.03
LID0.050.020.03
   Estradiol (pg/mL)*HID23.3–69.427.9–69.252.057.871.0
LID47.452.241.8
   Aldosterone (pg/mL)HID72.9–398.572.9–398.5ND169.0194.6
LIDND165.6192.0

Value for change in concentration from 0 to 12 months is significantly (P = 0.01) different between groups.

ND = Not determined.

Figure 1—
Figure 1—

Mean ± SEM serum concentrations of estradiol after ACTH administration in dogs that were evaluated before (0 months) and after (12 months) receiving an HID (n = 10) or LID (10) for 12 months. *Values at indicated time are significantly (P < 0.001) different.

Citation: American Journal of Veterinary Research 70, 3; 10.2460/ajvr.70.3.353

Isoflavone measurements—Daidzein, genistein, and glycitein concentrations were measured in serum and urine samples collected at 6 and 12 months to evaluate compliance of owners with feeding requirements and mean exposure of dogs to isoflavones. There was considerable variability in urine and serum isoflavone concentrations among dogs, which likely reflected the opportunistic timing of sample collection (ie, collection without specific regard to when dogs consumed their last meal). Nevertheless patterns in concentrations were detected (Table 3). Reflecting the dietary isoflavone content, concentrations of genistein, daidzein, and glycitein in serum and urine at 12 months were from 5 to 10 times as high in dogs in the HID group, compared with respective concentrations in dogs in the LID group. This difference in isoflavone content between diets was somewhat lower at 6 months after the diets were initiated, compared with that at 12 months after diets began.

Table 3—

Mean serum and urine concentrations of phytoestrogens measured in dogs that were evaluated at 6 months and 12 months after initiation of an HID (n = 10) or LID (10) for 12 months.

AnalyteSerum concentration (nmol/L)Urine concentration (nmol/mg of creatinine)
HIDLIDP value*HIDLIDP value*
Genistein
   6 mo166.4147.40.230.7440.1930.01
   12 mo222.775.71.1850.164
Daidzein
   6 mo246.768.30.0080.7390.2200.01
   12 mo254.47.91.0000.170
Glycitein
   6 mo91.700.020.5700.1740.008
   12 mo76.500.7540.084
Total isoflavones
   6 mo413.8215.70.031.9350.4130.004
   12 mo476.983.62.2550.333

Values of P < 0.05 were considered significant.

The HID had the following mean isoflavone content: 28 mg of daidzein/kg of dry weight, 68 mg of genistein/kg of dry weight, and 14 mg of glycitein/kg of dry weight. The LID had the following mean isoflavone content: 3 mg of daidzein/kg of dry weight, 4 mg of genistein/kg of dry weight, and < 2 mg (limit of quantification) of glycitein/kg of dry weight (Appendix). Estimated on the basis of mean food consumption by dogs, the daily intake of isoflavones in the HID group was 2.12 ± 0.25 mg/kg of body weight, and that in the LID group was 0.16 ± 0.11 mg/kg of body weight. The HID was more palatable than the LID. As the study progressed, 3 dog owners reported that their dogs were eating less and less of the assigned food; therefore, to allow them to complete the study, 2 of the dogs were served the food moistened with warm water, and 1 was served the food topped with a small amount of plain tomato sauce and grated parmesan cheese.

Discussion

In the study reported here, the effect of dietary phytoestrogens on endocrine status in dogs was evaluated. Our results indicated that feeding dogs a strictly soy-based diet for 1 year did not appear to have immediate adverse effects on their health. The results of the endocrine tests to evaluate function of the thyroid and adrenal glands, even considering the small number of dogs and the short duration of the study, suggested that changes in endocrine function might exist as reported for other species, but these changes need to be confirmed with clinical trials of longer duration.

The effect of phytoestrogens on thyroid gland function has been evaluated in laboratory, companion, and farm animals and humans. Studies in rats17 and cats7 fed a soy-based diet have revealed that serum fT4 and T4 concentrations increase without an apparent change in serum T3 concentration. On the other hand, results of another study18 involving rats indicated that serum TSH and T4 concentrations but not T3 concentrations decrease. It has been suggested that an association exists between consumption of soy formula in infancy and autoimmune thyroid gland disease in humans.19 In humans, ingestion of soy reportedly has no effect or reduces serum T3 concentrations but does not affect T4 concentrations20,21; however, the amount of soy protein to which humans are exposed in such studies is typically lower than that in animal studies. Increase in serum T4 concentrations relative to T3 concentrations could result from inhibition of the enzyme responsible for conversion of T4 to T3 (5'-iodothyronine deiodinase), as others have speculated.7 Although soy isoflavones have not yet been investigated as inhibitors of deiodinase, related flavonoid compounds in plants such as biochanin A, rutin, and quercetin are potent inhibitors, which can cause a 50% decrease in enzyme activity even at low micromolar concentrations.22,23

Deiodinase inhibition initially would be expected to decrease serum T3 concentrations. However, because T3 is the primary determinant of TSH release by the pituitary gland, homeostatic mechanisms should rapidly restore T3 concentrations nearly to within the reference range but with an increase in release of T4 by the thyroid gland to overcome enzyme inhibition. In the long term, there may be goiter formation and associated local hypermetabolism that may result in genetic mutations and feedback dysregulation (autonomous nodules lead to toxic nodular goiter), with an excessive increase in release of T4, leading to hyperthyroidism. A similar effect on serum concentrations of thyroid gland hormones (higher T4 concentration with unchanged T3 concentration) may also occur if ingestion of soy resulted in enhanced clearance of T3 (but not of T4) through induction of the enzymes responsible for metabolism of T3. In rats, inducers of T3 glucuronidation (eg, pregnenolone-16-α-carbonitrile) that result in thyroid hyperplasia have been identified.24

Estradiol exerts an effect directly on thyroid gland tissue through estrogen receptors,25 and the phytoestrogens can also bind to the estrogen receptors.26 Whether estrogen receptors that are capable of binding dietary phytoestrogens exist in thyroid glands of dogs is unknown. In our study, the results of thyroid hormone assays suggested that a small change in serum total T4 concentration may occur in dogs over a long period, but additional studies are necessary to explore this supposition.

Adrenocortical function may be altered by phytoestrogen intake because genistein and daidzein can inhibit the activity of 21-hydroxylase, which may lead to a decrease in cortisol synthesis and, as a consequence, an increase in synthesis of dehydroepiandrosterone and dehydroepiandrosterone-sulfate synthesis by shunting metabolites away from the glucocorticoid synthetic pathway.27 This hypothesis was not supported by the results of an in vivo study28 in which monkeys fed a soy-based diet had a lower adrenal weight and zona fasciculata thickness, compared with the same variables in a control group fed an isoflavone-depleted soy protein diet. It is unknown whether phytoestrogens have an effect on the enzymes involved in steroidogenesis in dogs.

Phytoestrogens are structurally and functionally similar to estradiol and have the ability to selectively bind estrogen B receptors more than estrogen A receptors.29 Phytoestrogens typically act as antiestrogens by competing with more potent endogenous estrogens for binding to estrogen receptors. In addition to the interaction with estrogen receptors, dietary phytoestrogens might compete with endogenous estrogens for the active site of the estrogenbiosynthesizing and estrogen-metabolizing enzymes and thus reduce the concentration of biologically active endogenous estrogens. Coumestrol and genistein reportedly inhibit the reduction of estrone to 17 β-estradiol by acting on 17 β-hydroxysteroid oxidoreductase type-1 enzyme.30 Genistein and daidzein also inhibit the enzymes 3 β-hydroxysteroid oxidoreductase and 17 β-hydroxysteroid oxidoreductase in vitro in human placental microsomes6 and in bovine adrenal glands.31

In the study reported here, no differences were evident between the HID and LID groups or within dogs among the 3 time points (0, 6, and 12 months) for any steroid hormones except estradiol. The differences between the 2 groups and within dogs in the HID group were significant, suggesting that phytoestrogens might have induced increases in serum concentrations of endogenous estrogens by blocking the estrogen receptors, thereby allowing more endogenous free estrogen to be measured. Clinical signs of hyperestrogenism (eg, hematologic abnormalities or coat changes) were not detected in affected dogs. One of the authors (JWO) has detected unusual signs of hyperestrogenism (polyuria and polydipsia, hepatomegaly, high serum activities of alkaline phosphatase and alanine aminotransferase, and dilute urine) in dogs with serum estradiol concentrations > 70 pg/mL. Seven of the dogs (6 males and 1 female) in the present study, of which 5 received an HID and 2 received an LID, had serum estradiol concentrations > 70 pg/mL; however, owners did not report polyuria or polydipsia, and only 3 dogs had activities of alkaline phosphatase, alanine aminotransferase, or both that were slightly higher than the upper reference limit at the end of the study. Cytologic evaluation of a vaginal swab sample to evaluate changes in mucosal cells in the only female with a high serum estradiol concentration was not performed.

Soy isoflavone concentrations were measured in serum and urine samples primarily to verify food intake of dogs and to evaluate uptake and excretion of phytoestrogen. As expected, mean serum and urine isoflavone concentrations reflected the difference in content of these compounds in the respective diets. However, concentrations were highly variable among dogs. This is likely the result of the opportunistic sampling protocol we used, which resulted in variation in sample collection time relative to the previous meal. Such opportunistic (or convenience) urine collection has been used extensively for evaluation of compliance of humans in soy-diet studies and yields good correlation between soy intake and urinary isoflavone concentration.32 Data are generally normalized to creatinine content to account for differences in urine output. Reported mean urinary concentrations of genistein, daidzein, and glycitein are 1.2 nmol/mg of creatinine, 1.9 nmol/mg of creatinine, and 0.6 nmol/mg of creatinine, respectively, for humans who ingest a soy-based diet,32 and these values compare well with those of the dogs that were fed the HID in the present study (mean values, 1.2 nmol/mg of creatinine, 1.0 nmol/mg of creatinine, and 0.8 nmol/mg of creatinine at 12 months). Serum isoflavone concentrations in the dogs in our study also compared well with values reported for people. For example, mean maximum plasma concentrations of genistein (410 nmol/L) and daidzein (470 nmol/L) are reported for humans that consumed a standardized soy beverage,33 whereas respective mean values were 223 nmol/L and 254 nmol/L for dogs that consumed the HID. Consequently, one may conclude that the degree of exposure of dogs to isoflavones in the present study was comparable to that of humans in other studies in which the health effects of soy were evaluated.

The only other report34 of serum isoflavone concentrations in dogs that could be identified concerns a toxicokinetic study of pure genistein administered orally to laboratory Beagles at dosages from 50 to 500 mg/kg/d. However, because these dosages are much higher than those used in our study, it is difficult to draw any direct comparisons. Indeed, the maximum reported serum genistein concentrations in those Beagles ranged from 4 to 11 μmol/L, depending on dosage, which is more than 10 times higher than concentrations detected in the present study. Interestingly, all Beagles in that study also received a standard canine laboratory animal diet that contained 7.8% vegetable protein. Furthermore, the serum total genistein concentration was 77 mg/kg of dry weight of food, which is slightly higher than the value for the HID used in the present study. Control dogs that received no additional genistein, apart from that contained in the food, were reported to have mean maximal serum genistein concentrations that ranged from 163 to 502 nmol/L, which is comparable to mean values for dogs that received the HID (166 nmol/L at 6 months and 223 nmol/L at 12 months).

A limitation of the present study is the sample of dogs used. It would have been ideal to have used dogs of the same breed, age, and sex, but a sample of mixed-breed dogs was enrolled because we believed it would be representative of our hospital population. Additional studies involving selected breeds should be carried out to evaluate the pharmacokinetic properties, bioavailability, and mode of metabolism of dietary phytoestrogens to determine whether differences exist among or within breeds of dogs. Our study also lacked standardization of dogs on the same diet prior to commencement. Such a standardization would have yielded values before the diet was administered that were comparable and would have ruled out biological differences caused by diets received prior to enrollment. Our study was not designed to evaluate the effect of soy on metabolism, but our aim was to maintain the body weight of dogs by adjusting their daily amount of food. Consumption of soy isoflavones reportedly increases metabolism, inhibiting lipogenesis and stimulating lipolysis, thus causing a decrease in body weight, adipose tissue deposition, and serum leptin concentration.35

Results of the study reported here suggested that dogs absorb and excrete phytoestrogens, and these phytoestrogens might have an effect on their hormonal status. As reported for other species of animals,36 some of the effects might be beneficial (eg, prevention of certain types of cancers or control of obesity), but there are also concerns about the estrogenic effect of consumption of high amounts of isoflavones, which, although weak, might have a biological effect when dogs receive such diets and treats on a long-term basis. To assess the potential risks and benefits of ingestion of soy isoflavones in puppies and adult dogs and the mechanisms by which health effects occur, it is essential to have a more complete understanding of the pharmacokinetics of isoflavones after consumption of soy foods. The impact of isoflavones on results of experiments carried out in animals to evaluate various health conditions should be considered when selecting a diet because some commercially available diets contain high isoflavone concentrations, which might have a biological effect.2,37 For certain types of experiments in endocrinology, a diet with no or low isoflavone content should be considered.

Abbreviations

fT4

Free thyroxine

HID

High-isoflavones diet

LID

Low-isoflavones diet

T3

Triiodothyronine

T4

Thyroxine

TSH

Thyroid-stimulating hormone

a.

Royal Canin Veterinary Diet Canine Hypoallergenic, Royal Canin, St Charles, Mo.

b.

Synacthen, Alliance, Chippenham, England.

c.

Sigma Chemical Co, St Louis, Mo.

d.

Deca XP Plus, Thermo Electron, Somerset, NJ.

e.

Synergi Fusion RP C-18 column, Phenomenex, Torrance, Calif.

f.

SAS, version 9.1, SAS Institute Inc, Cary, NC.

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Appendix

Content of diets used to study the effects of dietary soy isofla-vones in dogs.

IngredientHIDaLIDa
 Batch 1Batch 2Batch 1Batch 2
Moisture (%, as fed)4.77.95.36.4
Protein (%, as fed)23.922.723.423.6
Fat (%, as fed)20.818.120.417.1
Crude fiber (%, as fed)2.02.12.02.1
Ash (%, as fed)6.87.56.97.9
Caloric density (kcal/g)4.504.194.454.19
Genistein (mg/kg of dry wt)815562
Daidzein (mg/kg of dry wt)312624
Glycitein (mg/kg of dry wt)1711< LOD< LOD

Batch 1 was manufactured in August 2005, and batch 2 was manufactured in April 2006.

LOD = Limit of detection (2 mg/kg for glycitein). wt = Weight.

Contributor Notes

Supported by a grant from Royal Canin.

Dr. Court is supported by grants R01GM061834 and R21GM074369 from the National Institute of General Medical Sciences, National Institutes of Health.

The isoflavone-extracted hydrolyzed soy isolate was provided by The Solae Company.

The content of this report is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health.

The authors thank Joe Rogosky, Colleen Pinney, and Tara Miller for assistance with dermatologic evaluations and Qin Hao for assistance with isoflavone assays.

Address correspondence to Dr. Cerundolo.