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Original Studies |
Department of Internal Medicine, Cardioangiology, and Hepatology (P.F., F.M., B.N., G.C.), University Hospital S. Orsola-Malpighi, 40138 Bologna, Italy; Laboratory of Radioimmunology (L.P.), Rizzoli Orthopaedic Institute, 40136 Bologna, Italy; Laboratory for Biocompatibility Research on Implant Materials (L.S.), Rizzoli Orthopaedic Institute, 40136 Bologna, Italy; and Division of Geriatric Medicine (D.C.), University Hospital S. Orsola-Malpighi, 40138 Bologna, Italy
Address correspondence and requests for reprints to: Prof. Giovanni Ravaglia, Department of Internal Medicine, Cardioangiology, and Hepatology, University Hospital S. Orsola-Malpighi, Via Massarenti, 9, 40138 Bologna, Italy. E-mail: ravaglia{at}almadns.unibo.it
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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-tocopherol were studied in 44 healthy
Northern Italian oldest-old subjects (age range, 90–107 yr), selected
by the criteria of the SENIEUR protocol. Control groups included 44
healthy adult (age range, 20–65 yr) and 44 SENIEUR elderly (age range,
65–89 yr) subjects. Oldest-old subjects had higher TSH
(P < 0.01) and lower free T3
(FT3)/freeT4 (FT4) ratio,
zinc, and selenium serum values (P < 0.001)
than adult and elderly control subjects. No significant difference was
found for plasma retinol and -tocopherol values. The associations
between micronutrients and thyroid hormones were evaluated by
multivariate analysis. In oldest-old subjects, plasma retinol was
negatively associated with FT4 (P =
0.019) and TSH serum levels (P = 0.040), whereas
serum zinc was positively associated with serum FT3
(P = 0.010) and FT3/FT4
ratio (P = 0.011). In younger subjects, no
significant association was found among thyroid variables and
micronutrients. In conclusion, blood levels of specific micronutrients
are associated with serum iodothyronine levels in extreme aging. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Many authors (1, 4, 5, 6) suggest that these changes, rather than being caused by physiologic aging, are mostly secondary to coexisting chronic diseases or insufficient calorie intake, which can affect thyroid function at both the hypothalamic-pituitary regulation and peripheral tissue metabolism. Several studies have pointed out the risk of marginal deficiencies in vitamins and trace elements, even in seemingly well-nourished elderly populations living in industrialized countries (7, 8, 9).
In addition to iodine, several other micronutrients (10, 11, 12, 13) are involved in thyroid hormone metabolism, but only a few studies have addressed the possibility that marginal micronutrient deficits may contribute help to explain the alterations in thyroid function observed in advanced age (14, 15).
Therefore, the aim of this study was to evaluate the relationships between thyroid function and blood levels of selenium, zinc, retinol, and alpha-tocopherol in a selected group of free-living, healthy Italian subjects, 90 yr old and older.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The subjects were 65 free-living volunteers, 90 yr old or older, living in the urban area of Bologna, Emilia Romagna, Northern Italy, in January 1996. Details concerning recruitment and enrollment of participants, as well as assessment methods, have been published previously (16, 17). Briefly, to exclude the confounding effects of chronic or acute diseases on levels of circulating thyroid hormones, subjects were considered eligible for the study only if they fulfilled the health criteria selection of the SENIEUR protocol (18). This protocol describes the admission criteria for immunogerontological studies in man and is based on clinical, pharmacological, and laboratory data.1
Protein-calorie malnutrition, as defined by clinical judgment, is a SENIEUR exclusion criterion; and body mass index (BMI), calculated as weight (kilograms) divided by the square of the height (in meters), is the only anthropometric parameter specified as a guideline for malnutrition in the SENIEUR protocol (the minimal BMI required is 20 for females and 22 for males). For the purposes of this study, a nutritional assessment was done independently by two physicians trained in nutrition, on the basis of the subject’s clinical report, anthropometric measurements, and routine biochemistry. Anthropometric measurements were performed according to standardized procedures (19). All subjects were weighed on the same scales, barefoot and in light clothing. Because the spine’s shrinkage with aging can affect the validity of height measurement in the elderly, height was calculated from the knee-height measurement, according to the equations of Chumlea et al. (20). In addition to BMI, arm-muscle area (AMA) and arm-fat area (AFA) were also calculated for all subjects from mid-arm circumference and tricep skinfold thickness, using standard formulas (21). AMA and AFA values were compared with age- and sex-specific reference percentiles for Northern Italian nonagenarians and centenarians (19).
Additional exclusion criteria were: consumption of drugs known to affect thyroid function, and consumption of nutritional supplements.
Among the 65 oldest-old subjects enrolled in the study, 1 subject was found to have TSH levels less than 0.1 µUI/L, and 20 seemingly euthyroid subjects were found to have TSH levels more than 5 µUI/L and positive thyroid autoantibodies. These subjects were excluded from the analyses. This left 44 oldest-old subjects (31 women and 13 men, 90–107 yr old, of whom 24 were 100 yr old or older), who were compared with 44 elderly subjects (22 women and 22 men, 65–89 yr old) coming from the same area and selected according to the SENIEUR protocol, and 44 young healthy controls (22 women and 22 men, 20–64 yr old) recruited among the medical students and staff attending the Department of Internal Medicine, University of Bologna. All these subjects were euthyroid, and none had thyroid antibodies. Informed consent was obtained from all participants.
The 21 oldest-old subjects excluded from the analyses did not differ
significantly from the 44 included, with respect to age, sex, selenium,
zinc, retinol, and -tocopherol levels.
Blood measurements
Peripheral blood samples were collected, from 0800–0900 h, from overnight fasting subjects, put on ice, transported to the laboratories within 1 h, and processed immediately.
Plastic tubes containing tripotassium EDTA, and metal-free evacuated
tubes containing no additives (Becton Dickinson and Co.,
Meylan, France) were used for plasma and serum collection,
respectively. Plasma was separated by centrifugation (1500 x
g, for 30 min, at 4 C) and was immediately analyzed. Plasma
retinol and -tocopherol were assayed simultaneously by
reversed-phase high-performance liquid chromatography (model,
Millennium 2010; Waters Corp., Milford, MA) (22, 23). Coefficients of variations were less than 5% for both retinol and
-tocopherol.
Because plasma lipids influence blood concentrations of lipid-soluble
vitamins, plasma retinol and -tocopherol concentrations were
adjusted to plasma cholesterol and triglycerides levels (24).
Serum was separated by centrifugation (3000 x g, for 30 min, at 4 C), and aliquots were appropriately stored at -70 C until analyzed.
Serum free T3 (FT3), free T4 (FT4), and TSH were assayed by commercial fluoroenzymatic kits (Eurogenetics, Tessenderlo, Belgium). Intraassay and interassay coefficients of variation were 4.7% and 7.4% for TSH, 6.4% and 7.1% for FT3, and 6.9% and 4.2% for FT4. Normal ranges for our laboratory were 0.2–5 µUI/L for TSH, 2.2–6.9 pmol/L for FT3, and 10.3–25.7 pmol/L for FT4. Autoantibodies to thyroglobulin and thyroid peroxidase were assayed by immunoradiometric assay (Anti-HGT Bridge; Biodata Guidonia Montecelio, Rome, Italy: intraassay and interassay coefficients of variation were 5% and 6.3%; TPO kit, BIO-LINE, Brussels, Belgium: intraassay and interassay coefficients of variation were 4% and 5.5%). Antibody values above 100 UI/mL were considered positive.
Serum selenium concentrations were determined in triplicate, with a graphite furnace atomic absorption spectrometer with the Zeeman background correction (model, Solaar 939QZ; UNICAM, Cambridge, UK), by using a standard addition method. Bovine serum with a low selenium concentration was used to prepare the standard curve. Human sera (standard reference material 1598, National Institute of Standards and Technology, Gaithersburg, MD) were used for validating the accuracy and precision of the method. Samples were compared with the standard curve by using the standard curve, linear least-squares fit analysis. The detection limit for selenium was 0.093 µmol/L (7.4 ng/mL).
Serum zinc concentration was determined using a flame atomic absorption spectrometer (model Pye Unicam PU9400; Philips, Eindhoven, The Netherlands) equipped with an air-acetylene flame burner. A linear calibration curve was performed by using certified standard National Institute of Standards and Technology solutions at three concentrations (0.1, 0.2, and 0.3 mg/L). Specimens were diluted 1:5 with 5% glycerol ultrapure bidistilled and deionized water and analyzed in duplicate. Seronorm Trace Elements (Nycomed Pharma, Oslo, Norway) and Serum Trace Elements Control Toxicology, Normal Range, (Utak Laboratories Inc., Valencia, CA), were used as controls for validating method accuracy and precision. The detection limit for zinc was 0.153 µmol/liter (0.01 µg/mL).
Within-run and run-to-run coefficients of variations were 1.7 and 4% for selenium and 2.5% and 5.3% for zinc.
Statistical methods
Data are reported as mean ± SD. Because the distributions of values for TSH and plasma retinol were markedly skewed, these variables were natural-log transformed before analysis and reported as geometric mean. The 95% confidence interval of the geometric mean was found by taking the antilog of the 95%-confidence interval of the log-transformed variables. Overall analysis of the hormonal and nutritional variables in the three study groups was performed by ANOVA. When ANOVA indicated significant differences between groups (P < 0.05), all-pairwise post hoc comparisons were made by Bonferroni’s test.
Univariate relationships were studied by Pearson product-moment correlation. The relative strength and interdependence of these relationships were investigated by forward stepwise regression.
Statistical calculations were performed using SYSTAT software (SYSTAT 8.0; SPSS, Inc., Chicago, IL).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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, oldest-old
subjects had normal biochemical indices of nutrition. Their
anthropometric indices, although largely below the average values for
younger people, were also normal, according to sex-specific reference
values for Northern Italian nonagenarians and centenarians (19).
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, serum TSH levels
were significantly higher in oldest-old than in young and elderly
subjects. FT4 levels did not significantly differ
among groups. No individual result for FT3 in the
oldest-old group was below the given reference ranges; yet, average
FT3 levels were significantly lower in this age
group than in young and elderly subjects. The
FT3/FT4 ratio was also
significantly different among groups, with oldest-old subjects having
the lowest FT3/FT4 ratio.
To exclude that mild thyroid autoimmune disease could influence TSH and
FT3 serum levels, analyses were also performed
after excluding sera of oldest-old subjects positive for antithyroid
antibody (four subjects). After exclusion of these samples, the
differences in TSH, serum FT3, and
FT3/FT4 ratio values among
the study groups were fully confirmed.
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-tocopherol plasma
levels. Because young and elderly subjects did not differ with regard to thyroid function and micronutrients values, for the following statistical analyses, we pooled them in one group to be compared with oldest-old subjects.
Tables 3 and 4 summarize the results for univariate
correlation analysis between thyroid hormones and micronutrient blood
concentrations. In subjects below 90 yr of age (Table 3),
FT3 and FT4 serum levels
were significantly correlated with each other. In the same subjects,
FT3 levels also significantly correlated with
serum zinc and plasma retinol levels, and serum
FT4 levels significantly correlated with serum
selenium levels. In addition to the obvious significant correlations
with both FT3 and FT4, the
FT3/FT4 ratio also
significantly correlated with serum zinc but not with serum selenium or
plasma retinol levels.
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In oldest-old subjects, but not in younger subjects, zinc and selenium serum levels were highly interrelated (r = 0.443, P = 0.004).
For both oldest-old and younger subjects, results did not significantly differ when subdividing subjects by sex (data not shown).
The relative strengths and interdependence of the univariate relationships were investigated by forward stepwise regression in younger and oldest-old subjects separately. Stepwise multiple regression for each thyroid hormone was performed, including age, sex, and all the other thyroid variables (the FT3/FT4 ratio model obviously included TSH only).
In subjects below 90 yr of age (Table 5), stepwise results confirmed the
interrelationships among FT3,
FT4, and TSH serum levels. A significant effect
of sex was also found, with men having slightly lower TSH values than
women. Stepwise regression also identified a significant positive
association of age with serum FT4 levels not
identified by ANOVA. None of the associations between iodothyronine and
micronutrient blood levels found in univariate analysis was
confirmed.
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),
stepwise analysis showed that both TSH and FT4
serum levels were negatively associated with age and plasma retinol
levels, whereas serum FT3 levels and the
FT3/FT4 ratio were
positively associated with serum zinc values. Stepwise analysis results
did not change when including a Se*Zn interaction term.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The major strength of this study is the particular care devoted to the selection of the study subjects. Serum levels of thyroid hormones can be affected by a great variety of drugs and acute or chronic diseases, including nutritional problems (1). Therefore, we adopted the very strict exclusion criteria of the SENIEUR protocol, to avoid that possible changes in thyroid hormone concentrations might reflect diseases associated with age rather than being an effect of aging per se. Because the SENIEUR criteria for exclusion of protein calorie malnutrition are mainly clinical, an additional strength of this study is the availability of objective measures of nutritional status, such as anthropometric and laboratory parameters, for the oldest-old group.
However, this study has also several limitations. First, dietary intake of energy and nutrients was not evaluated, although it would have been very useful, to thoroughly characterize nutritional status and explain the reason of the low selenium and zinc blood levels of oldest-old subjects. However, all the nonagenarians and centenarians included in this study were free-living; and, when attempting to collect diet records, we could not obtain a reliable estimate of portion sizes. Thus, actual amounts of ingested food could not be quantified.
Second, unmeasured factors, such as iodine dietary intake, may have confounded the study results. However, the urban area of Bologna is not an endemic goiter area, and iodine intake in this area has been reported to be generally adequate (25). Moreover, subthreshold concentrations of iodine intake are associated with a variety of alterations in thyroid function indicators, including an increased serum T3/T4 ratio and decreased FT4 concentrations (1), which were not found in our study sample. A further important limitation of this study is its cross-sectional study design, which does not permit us to ascertain whether the alterations in micronutrient blood levels are the cause or the effect of the impaired thyroid function.
In the current study, thyroid function seemed to be well preserved until the eighth decade of life, whereas a reduction of serum FT3 levels was observed in extreme aging. Several studies have shown that, when care is taken to select elderly subjects without concomitant nonthyroidal diseases, advanced age per se is not accompanied by alterations in thyroid hormone concentrations (4, 5). These studies, however, included only small numbers of people over 85 yr of age.
In contrast with these studies, but in agreement with our results, Mariotti et al. (3) reported a decrease in serum FT3, along with unchanged serum FT4 levels, in a relatively large sample of SENIEUR centenarians. In their population, however, serum TSH levels of centenarians were found to be decreased, whereas we observed an increase in serum TSH levels of oldest-old subjects. The study selection protocol was the same for both our and the Mariotti et al. study (3), but a possible reason for this discrepancy may be the relatively younger age of our study group, composed, in almost equal parts, by people in their 9th and 10th decades. We carefully excluded subjects with serum TSH concentrations out of the normal range, and the increased TSH levels of oldest-old subjects were confirmed, even after exclusion of subjects positive for thyroid autoantibodies. It must not be overlooked, however, that the prevalence of thyroid autoantibodies is reduced in the extreme decades of life (26) and that most cases of subclinical hypothyroidism among old-subjects living in iodine-rich regions have been reported to be not of autoimmune origin (27). Because thyroid morphology was not studied, thyroid dysfunction in the oldest-old group may have been undervalued.
The lack of interrelationships between TSH and iodothyronines found in this age group supports the hypothesis of a derangement in the central regulation of TSH secretion (3, 28). On the other hand, however, the significant decrease in the FT3/FT4 ratio with age also points to a decline in peripheral 5'deiodination of circulating T4 to T3. The careful exclusion of subjects with clinical and biochemical signs of overt systemic disease or chronic malnutrition argues against a classical euthyroid sick syndrome (1). However, a low T3 syndrome has been reported in association with low carbohydrate intake (29) and low calorie diet without clinical or biological evidence of malnutrition (30). Because, at the study time, calorie intake and diet composition were not investigated, these conditions cannot be entirely ruled out.
To our knowledge, only a few studies have been published about the relationships between thyroid function and specific trace elements, in relation to the aging process (14, 15); and information on the relationships between vitamin status and age-related changes in thyroid function is virtually absent.
Selenium and zinc have important roles in thyroid metabolism (12, 13), and a decrease in their serum levels is thought to be common in human aging (31).
Selenocysteine has been identified in the active center of types I and III iodothyronine deiodinase (12, 32). Selenium is also incorporated into glutathione peroxidase, which plays a fundamental role in the thyroid defenses against the oxygen-derived free radicals normally produced in thyrocytes during thyroid hormone synthesis (33).
Zinc has a fundamental role in protein synthesis (13), is involved in T3 binding to its nuclear receptor (34), and participates in the formation and mechanism of action of TRH (35). Lowered serum zinc concentrations have been described in untreated hypothyroid patients (36).
In agreement with data from animal experiments (37), Olivieri et al. (14, 15) reported that, in euthyroid elderly people, a poor selenium status was associated with a diminished 5' deiodination, leading to increased T4 levels and decreased T3/T4 ratios. In the same population, however, thyroid hormones did not correlate with indices of zinc status; although, in both rats (37, 38) and humans (39), zinc deficiency has been reported to decrease iodothyronine levels.
In the current study, in agreement with the findings of Olivieri et al. (15), oldest-old subjects had both zinc and selenium serum levels decreased, with respect to young and elderly controls. In oldest-old subjects, however, no relationship was found between selenium and iodothyronine levels, whereas we found a strong positive association between zinc and FT3 levels.
This agrees with results from Hagmar et al. (40), who, in a recent study of Latvian fish consumers, did not observe any impact of selenium status on thyroid parameters, except for a slight negative correlation of selenium status with serum TSH levels.
In a study of patients with euthyroid sick syndrome (41), both selenium and serum T3 levels were found to be decreased. No independent relationship, however, was found, in this study, between the extent of the low T3 syndrome (as manifested either as the absolute serum T3 concentration or the T3/T4 ratio) and serum selenium concentration; whereas they both seemed to reflect the changes in the degree of disease severity.
The lack of effect of marginal selenium deficiency on iodothyronine levels in humans may have several explanations. First, in selenium deficiency, this trace element is relatively well maintained by the thyroid, with type I iodothyronine deiodinase having a preferential supply of selenium over glutathione peroxidase (32). This could be the reason that only in very severe selenium deficiency, such as found in China, will abnormal thyroid function tests be observed in humans (42). Moreover, animal experiments suggest that, during long-term selenium deficiency, the hypothalamic-pituitary axis could reset to normalize TSH and T3 concentrations, in spite of the increased T4 levels and the reduced peripheral deiodation (42, 43). Finally, whereas both selenium and zinc deficiency interact with iodine deficiency, selenium deficiency does not seem to enhance the effect of zinc deficiency on circulating iodothyronine in non-iodine-deficient rats (38).
Although serum zinc concentration is not a reliable index of zinc status (44), the positive association found in this study between FT3 and zinc blood levels supports the hypothesis that a lower zinc availability has a role in the decreased serum FT3 levels of older subjects. However, thyroid hormones also seem to modulate intestinal and renal zinc transport (45). If so, the reduced thyroid function of older people would be a cause, and not an effect, of the reduced zinc blood levels. Unfortunately, it is not for the cross-sectional design of this study to solve this question.
We also found an inverse association of both TSH and FT4 serum levels with plasma retinol levels of oldest-old subjects. Explanations of this finding can only be speculative, because the relationships between retinol and thyroid function are very complex and not yet fully understood. Overt hypothyroidism may increase retinol levels by reducing its hepatic uptake (11). However, no relationship between thyroid function and plasma retinol levels was found in our younger controls, although their plasma retinol levels were similar to those of the oldest-old. Therefore, the reduced thyroid function found in extreme aging could be a cause, rather than an effect, of disturbances in retinol metabolism. In fact, recent experimental evidence in rats suggests that retinoic acid, an active metabolite of retinol, may inhibit TSH secretion (46). Then, an enhanced inhibitory effect of retinol on thyroid function might contribute toward the general disruption of the pituitary-hypothalamic-thyroid axis observed with aging.
A number of reports also suggest a relationship between -tocopherol
and thyroid function (47, 48), but no association was found between
thyroid hormones and plasma -tocopherol levels in the current
study.
In conclusion, this study supports the hypothesis that extreme aging is characterized by a complex derangement in thyroid function. It also demonstrates that blood levels of some micronutrients are related to thyroid function in oldest-old people. Further investigations, however, are needed, to better clarify the physiologic importance of these findings and to evaluate the effects and risks of micronutrient supplementations for thyroid function in older people.
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Footnotes |
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Received August 5, 1999.
Revised December 29, 1999.
Revised March 16, 2000.
Accepted February 22, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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