Discrepancia en concentraciones de hormonas tiroideas libres medidas por distintos inmunoensayos / Discrepancy in concentrations of free thyroid hormones measured by different immunoassays

Abstract: Thyroid function is assessed by measuring thyrotropin and free and total thyroid hormone concentrations. There are interferences with the results of immunoassays that can lead to an incorrect diagnosis, of which the most frequent are the binding of thyroid hormones to heterophile antibodies, rheumatoid factor, anti-Ruthenium antibodies, the intake of biotin and anti-streptavidin antibodies. We present three cases of clinically euthyroid patients, with normal TSH, high free T4 and T3, and normal total T4 and T3 performed in a Roche Diagnostics ® COBAS 8000 device. When the test was repeated on a Siemens® Immulite device, the free and total hormones were within normal ranges. In the Roche Diagnostics ® assay, the presence of biotin or anti-Ruthenium or anti-streptavidin antibodies interferes with the formation of the complex responsible for the emission of light that allows inferring concentrations of thyroid hormones. The Siemens test works differently since the emission of light depends on the binding of T4 to an antibody conjugated with alkaline phosphatase not participating in the process biotin, streptavidin or ruthenium so this interference is avoided. This possible interference in immunoassays should be taken into account in case clinical manifestations differ from these laboratory determinations, to avoid a diagnosis and potential inappropriate treatment.

Resumen: La función tiroidea se evalúa midiendo tirotropina y concentraciones de hormonas tiroideas libres y totales. Existen interferencias con los resultados de inmunoensayos que pueden llevar a un diagnóstico incorrecto, de ellas, las más frecuentes son la unión de hormonas tiroideas a anticuerpos heterófilos, el factor reumatoide, anticuerpos anti Rutenio, la ingesta de biotina y anticuerpos anti estreptavidina. Se presentan tres casos de pacientes clínicamente eutiroideos, con TSH normal, T4 y T3 libres elevadas, y T4 y T3 totales normales realizadas en un equipo COBAS 8000 de Roche Diagnostics®. Cuando se repitió el ensayo en un equipo Immulite de Siemens®, las hormonas libres y totales estaban dentro de rangos normales. En el ensayo de Roche Diagnostics ®, la presencia de biotina o anticuerpos anti Rutenio o anti estreptavidina, interfiere con la formación del complejo responsable de la emisión de luz que permite inferir las concentraciones de las hormonas tiroideas. El ensayo de Siemens funciona de manera diferente ya que la emisión de luz depende de la unión de la T4 a un anticuerpo conjugado con fosfatasa alcalina no participando en el proceso biotina, estreptavidina o Rutenio por lo que se evita esta interferencia. Esta posible interferencia en inmunoensayos debe ser tenida en cuenta en caso de que las manifestaciones clínicas difieran de estas determinaciones de laboratorio, para evitar un diagnóstico y potencial tratamiento inadecuado.

Estudio e identificación de microRNA en exosomas urinarios en hipertensión arterial mineralocorticoide / Study and identification of microRNA in urinary exosomes in mineralocorticoid arterial hypertension

About 15% of the essential hypertensive patients would have a low activity of the 11ßHSD2 enzyme, which inactivates cortisol (F) to cortisone (E). Gene expression can be negatively regulated by miRNA. Urinary exosomes and their specific content (miRNA/proteins) represent a valuable tool as a biomarker for the diagnosis and prognosis of the disease. Aim: To evaluate the expression of miRNA specific for 11ßHSD2 in samples of urinary exosomes and to determine its association with biochemical variables associated with mineralocorticoid metabolism. Subjects and Methods: Cross-sectional study in subjects between 10-60 years. They were classified into subjects with high F/E (> p75) and low cortisone (< p25) and control subjects. The urinary exosomes were isolated with the Invitrogen kit. Bioinformatic analysis was performed with Mir Walk to identify specific miRNAs of HSD11B2. The expression of miRNA was evaluated by qRT PCR. The comparisons were made with the Mann-Whitney test. Results: 7.1% of the subjects are suggestive of a partial deficiency of 11ßHSD2 (NC-AME). The expression of miR-488 was higher in NC-AME than in controls (5839 ± 1719 vs 3,437 ± 2,581; p = 0.01). We found positive associations between mir-615 and ARP; miR-488 and the sodium/potassium ratio; miR-1205 with age and urinary sodium excretion; miR-494 with age, activity MMP9 and NGAL. Conclusion: We identified high expression of miR488 in NC-AME subjects and associations of miRNAs with biochemical variables associated with mineralocorticoid metabolism. Thus, exosomes and their miRNA content could be potential regulators and biomarkers of 11ßHSD2 activity.

Aldosterona e IL-17 en la génesis de la hipertensión arterial mineralocorticoídea, un estudio ex vivo / Aldosterone and il-17 in the genesis of mineralocorticoid arterial hypertension: an ex vivo study

Background: Treatment of dendritic cells (DC) with aldosterone induces the secretion of IL-6 and TGF-beta. The polarization of naïve T cells to helper 17 T lymphocytes with DCs pre-incubated with aldosterone, has been described in vivo, generating an IL-17 hyper-secreting phenotype, a cytokine associated with cardiac and renal fibrosis. There are mineralocorticoid receptors (MR) in immune cells and their activation may determine the inflammatory (M1) or adaptive (M2) macrophage phenotype. Aldosterone levels could regulate immunogenic gene expression in these cells, modulating the liberation of specific cytokines. Aim: To assess in humans the association of aldosterone levels and IL-17 with inflammatory markers in peripheral blood mononuclear cells (PBMC). Material and Methods: In blood samples of 176 participants aged 18 to 67 years (61 percent women) with a body mass index of 27.1 +/- 4.8 kg/m2, aldosterone, plasma renin activity (ARP), cortisol, C reactive protein, andIL-17 were measured. mRNA was isolated from PBMCs to measure the expression of MR RAC-1, HO-1, TLR-4, CD-14, NGAL and IL-17 by real time polymerase chain reaction. Results: Aldosterone correlated positively with ARP and the expression of CD-14 in PBMCs. Plasma levels of IL-17 were positively associated with the expression of MR, Rac1a and NGAL. Conclusions: Aldosterone and IL-17 levels were associated with inflammatory activation markers in PBMC, which could activate MRand promote a subclinical inflammatory status inducing hypertension.

Alta frecuencia de patología tiroidea funcional y autoinmune en mujeres embarazadas chilenas catalogadas como sanas / High frequency of functional and autoimmune thyroid disease in women classified as healthy Chilean pregnant

Background: Untreated functional thyroid diseases are a risk factor for maternal and fetal complications during pregnancy. Aim: To determine the frequency of functional or autoimmune thyroid disease in healthy women during the first trimester of pregnancy. Subjets and Methods: healthy pregnant women attending a routine consult during their first trimester of pregnancy were studied. Thyroid stimulating hormone (TSH), total and free thyroxin (T4) anti-thyroid peroxidase (TPO) antibodies and spot urine iodine levels were measured. The reference ranges provided by the Atlanta Georgia Consensus in 2004 were used as normal values. A urine iodine concentration < 150 ug/L was considered low. Results: One hundred women age 30 +/- 5 years with a mean gestational age of 8,8 +/- 1,9 weeks, were studied. The frequencies of subclinical hypothyroidism, clinical hypothyroidism, isolated low thyroxin lecels, high antiTPO antibodies and low urine iodine levels were 19, 2, 3, 13 and 15 percent, respectively. Women with high TSH levels had lower total and free T4 levels. Conclusions: Twenty one percent of this sample of apparently healthy pregnant women had a clinical or subclinical hypothyroidism.

[Assessment of androgenic decline in the elderly]. / Comparación de distintos métodos para evaluar la función androgénica en el adulto mayor.

BACKGROUND: The gold standard to assess androgenic status is the measurement of free testosterone by equilibrium dialysis. However, the estimation of free testosterone using formulas based on the law of mass action can be an adequate standard. AIM: To assess androgenic decline in the elderly by different methods. MATERIAL AND METHODS: Free testosterone by radioimmunoassay, total testosterone and steroid binding globulin (SHBG) by automated chemiluminiscence system and estradiol by automated electrochemiluminiscence system were measured in 30 male aged 64+/-5 years (range 60-70), and 25 males aged between 20 and 30 years, as control group; devoid of diseases or drugs that could cause hypogonadism. Free androgen index, free testosterone, biavailable testosterone, and free estradiol were calculated using a formula based on the law of mass action. RESULTS: Fifty seven percent of elderly subjects had hypogonadism, according to calculated free testosterone values. Their total testosterone was on average, 152 nd/dl lower than in young adults, figure that represents a 3.8 ng/dl decline per year. According to total testosterone values, 27% of elderly males had gonadal incompetence. The correlations between calculated free testosterone and total testosterone was 0,95 and between calculated free testosterone and measured free testosterone was 0,67. The methods employed overestimated (76.7% of hypogonadism when using the free androgen index) or underestimated (27 and 3% of hypogonadism, considering total or free testosterone, respectively) the ondrogen decline of the elderly. Among the elderly, 16 or 30% of subjects had an absolute hypoestrogenism, based on estradiol or calculated free estradiol values, respectively. On average there was a 20 and 30% reduction of estradiol and calculated free estradiol values in the elderly. CONCLUSIONS: Calculated free or bioavailable testosterone values should be used to assess androgen decline in elderly men.

Comparación de distintos métodos para evaluar la función androgénica en el adulto mayor / Assessment of androgenic decline in the elderly

Background:The gold standard to assess androgenic status is the measurement of free testosterone by equilibrium dialysis. However, the estimation of free testosterone using formulas based on the law of mass action can be an adequate standard. Aim: To assess androgenic decline in the elderly by different methods. Material and methods: Free testosterone by radioimmunoassay, total testosterone and steroid binding globulin (SHBG) by automated chemiluminiscence system and estradiol by automated electrochemiluminiscence system were measured in 30 male aged 64±5 years (range 60-70), and 25 males aged between 20 and 30 years, as control group; devoid of diseases or drugs that could cause hypogonadism. Free androgen index, free testosterone, biavailable testosterone, and free estradiol were calculated using a formula based on the law of mass action. Results: Fifty seven percent of elderly subjects had hypogonadism, according to calculated free testosterone values. Their total testosterone was on average, 152 nd/dl lower than in young adults, figure that represents a 3.8 ng/dl decline per year. According to total testosterone values, 27 percent of elderly males had gonadal incompetence. The correlations between calculated free testosterone and total testosterone was 0,95 and between calculated free testosterone and measured free testosterone was 0,67. The methods employed overestimated (76.7 percent of hypogonadism when using the free androgen index) or underestimated (27 and 3 percent of hypogonadism, considering total or free testosterone, respectively) the ondrogen decline of the elderly. Among the elderly, 16 or 30 percent of subjects had an absolute hypoestrogenism, based on estradiol or calculated free estradiol values, respectively. On average there was a 20 and 30 percent reduction of estradiol and calculated free estradiol values in the elderly. Conclusions: Calculated free or bioavailable testosterone values should be used to assess androgen decline in elderly men.

Actividad de 11 beta hidroxiesteroide dehidrogenasa tipo 2 en hipertensos chilenos / 11 B-hydroxysteroid dehydrogenase activity in patients with hypertension and low plasma renin activity

Background: Half of hypertensive patients with low plasma renin activity have a primary hyperaldosteronism. Among the remaining half, 11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2) deficiency plays an important role. This enzyme catalyzes the conversion of cortisol to cortisone, avoiding the interaction of cortisol with the mineralocorticoid receptor. If the enzyme fails, cortisol will stimulate sodium and water reabsorption and increase blood pressure. Aim: To determine biochemical alterations, suggestive of 11ßHSD2 deficiency, in low-renin hypertensive patients. Patients and Methods: Twenty eight hypertensive patients with a plasma renin activity of less than 0.5 ng/ml/h and with a plasma aldosterone of less than 5 ng/dl were studied. Twenty eight normotensive patients were studied as controls. Serum cortisol (RIA), cortisone (ELISA) and the serum cortisol/cortisone ratio were determined in all of them, between 9 and 10 AM. Measurements were confirmed by high pressure liquid chromatography. The serum cortisol/cortisone ratio was considered abnormal when its Ln (cortisol/cortisone) value was over 2 standard deviations of the mean. Results: Serum cortisol was higher in hypertensive subjects than in controls (11.1ñ3.3 and 9.2ñ2.8 µg/dl, respectively; p <0.05). No differences were observed in serum cortisone (3.4ñ1.3 and 3.7ñ1.2 µg/dl, respectively). Four hypertensive subjects had an abnormally high Ln (cortisol/cortisone) value (1.86; 1.73; 2.07 and 2.01, considering a normal value of less than 1.61). Conclusions: Four of 28 hypertensive subjects with low plasma renin activity and aldosterone had biochemical alterations suggestive of 11ßHSD2 deficiency

Anticuerpos anti-tiroglobulina en el seguimiento de pacientes con cáncer diferenciado de tiroides: ¿marcadores de enfermedad residual o recidivante? / Anti thyroglobulin antibodies in the follow up of patients with differentiated thyroid cancer

Background: Anti thyroglobulin antibodies are present in 25 percent of patients treated for a differentiated thyroid cancer, invalidating thyroglobulin determination. Those patients subjected to total thyroidectomy and free of disease, should reduce the production of these antibodies, due to the lack of antigenic stimulus. Therefore, anti thyroglobulin antibodies could be useful to detect early relapses. Aim: To assess the relationship between anti thyroglobulin antibodies and the evolution of the disease in patients treated for thyroid cancer. Material and methods: Retrospective analysis of 26 patients treated for thyroid cancer with positive anti thyroglobulin antibodies, followed for three years. These were divided in those with or without lymphocytic thyroiditis (19 and 7 respectively). Results: At the first year of follow up, anti thyroglobulin antibody concentration was 401ñ94.9 UI/ml (xñsem) in patients with thyroiditis and 38.9ñ8.9 UI/ml in those without thyroiditis (p < 0.005). During the three years of follow up, no differences in anti thyroglobulin antibodies were observed between patients with or without tumor relapse. Conclusions: Concentration of anti thyroglobulin antibodies was higher in patients with thyroiditis and did not differentiate patients with tumor relapse

Efecto de dosis bajas de calcitonina sobre la remodelación ósea en mujeres postmenopáusicas con alto recambio / Effects of low calcitonin doses on bone remodeling in postmenopausal women with high bone turnover rate

Background: Calcitonin is specially indicated for the treatment of osteoporosis in women that cannot receive estrogen replacement therapy or that have a high bone turnover rate. Aim: To study the effects of low intranasal calcitonin doses on bone remodeling in postmenopausal women with a high bone turnover. Patients and methods: Forty one healthy women aged 56 ñ 6 years old, with a mean lapse after menopause of 7.6 ñ 6.5 years and with a high bone turnover rate, evidenced by an urinary hydroxyproline (mg/dl)/creatinine (g/dl) ratio of 52.4 ñ 7.2, were studied. They were randomly assigned to receive 100 or 50 U/calcitonin thrice a week during 3 months or to a control group that received placebo. All received 500 mg/day calcium carbonate. Urinary hydroxyproline/creatinine ratio was measured a 0, 15, 30, 60 and 90 days. Plasma bone fraction of alkanine phosphatases was measured at 0, 30 and 90 days. Results: Initial urinary hydroxyproline/creatinine ratio and plasma bone fraction of alkanine phosphatases were similar in all study groups and there was no change in these parameters during the study period. Conclusions: Intranasal calcitonin in doses of 100 U thrice a week or less, does not modify accelerated bone turnover in postmenopausal women

Autoanticuerpos anti-tiroglobulina: detección y posible interferencia en la medición de tiroglobulina en suero / Thyroglobulin autoantibodies: detection and possible interference in serum thyroglobulin measurement

Background: Thyroglobulin measurement is useful for the follow up of patients subjected to total thyroidectomy for differentiated thyroid carcinoma. Thyroglobulin autoantibodies may interfere with its determination. Aim: To measure thyroglobulin autoantibodies and their interference with thyroglobulin determination. Material and methods: The presence of thyroglobulin autoantibodies was investigated in 801 serum samples sent to the laboratory for measurement of thyroglobulin levels. A serum was considered positive for these autoantibodies when radioactivity corresponding to 125I-thyroglobulin bound to thyroglobulin autoantibodies, precipitated with human gamma globulin, exceeded in 1.4 times that of a negative sera pool. In positive sera, thyroglobulin autoantibody concentration was measured and its interference with thyroglobulin radioimmunoassay was assessed through a recuperation test using exogenous thyroglobulin. Results: Thyroglobulin autoantibodies were detected in 149 sera (18.6 percent). Of these, 65 had a recuperation that fluctuated between 1 and 80 percent. Thyroglobulin autoantibody concentration was negatively correlated with recuperation percentages (r= -0.64; p <0.001) but not with thyroglobulin concentrations (r= 0.08). Thyroglobulin was higher in positive sera with a recuperation over 80 percent than in sera with a recuperation of less than 80 percent (12.7 ñ 1.7 and 5.9 ñ 0.6 ng/ml, respectively; p <0.001). Conclusions: Thyroglobulin autoantibodies interfere with thyroglobulin measurement by radioimmunoassay, sequestering variable amounts of thyroglobulin. The presence of these autoantibodies must be investigated prior to thyroglobulin determination

Hipertiroxinemia con eutiroidismo clínico: análisis a propósito de un caso / Hyperthyroxinemia and clinical euthyroidism: report of one case

The association of hyperthyroxinemia and euthyroidism is frequent and characterized by high plasma thyroxin concentrations, normal TSH values and absence of clinical signs of hyperthyroidism. We report an asymptomatic 28 years old male presenting with a serum total plasma thyroxin of 18.5 µg/dl (N 6.1-12.5), a free thyroxin of 2.9 ng/dl (N 0.8-1.4), a TSH of 3.4 µIU/ml (N 0.5-5), and a triiodothyronine of 128 ng/dl (N 80-180). Laboratory assessment did not find high thyroxin binding globulin, albumin or prealbumin concentrations or antithyroxin antibodies. The thyroxin binding capacity of albumin was elevated to 58.2 µg/dl (N 11.5-34.1). TSH responded normally to TRH stimulus and was suppressed with exogenous triiodothyronine, which caused an hyperthyroid syndrome. We concluded that this patient had a familial dysalbuminemia

Velocidad de disolución de diferentes preparados de calcio utilizados en clínica / Dissolution velocity of different calcium solutions used in clinics

Hydrochloric acid was added to destilled water in increasing amounts to obtain a final pH of 6.9, 3.0, 2.5, 2.0 and 1.5. Eighteen commercial calcium preparations were incubated in these solutions for 60 min and dissolution velocity was measured as the percentage of elemental calcium found in solution after this incubation period. Calcium carbonate preparations had a pH 1.5. Using the solution with pH 1.5 the dissolution velocity of different preparations varied widely from 56 to 100 percent. Calcium acetate, followed by calcium citrate and dicalcic phosphate were the salts in tablets with better dissolution velocities. Among powders and effervescent preparations, those containing calcium lactogluconate and citrate had the better dissolution velocities (95 to 115 percent), that were independent of the solution's pH. A studied preparation with integral bone had a very low dissolution velocity, not surpassing 33 mg of calcium per tablet. The dissolution velocity of different calcium carbonate preparations varies greatly and in conditions of achlorhydria, it is negligible. Calcium lactogluconate and citrate dissolution velocities are independent of the solution's pH

Importancia del déficit de inhibidores de la cristalización en la etiopatogenia de la urolitiasis / Importance of crystallization inhibitors deficit in the etiopathogenic of urolithiasis

Background: five percent of consultations at the emergency room of Catholic University Hospital are due to nephrolithiasis. The causes of this high frequency remain unknown. Aim: to know the main metabolic and anatomic factors involved in the genesis of nephrolithiasis. Patients and methods: 41 patients (31 male) were studied presenting with a renal colic were studied as soon as the acute episode subsided and without diet modifications. Fasting blood calcium and creatinine and 24 h urine calcium, uric acid, citrate, magnesium and pH were measured and an intravenous pyelogram was performed. 21 subjects without a history of nephrolithiasis were used as controls. Results: Patients with nephrolithiasis did not differ from controls in urinary calcium (159 ñ 67 and 172 ñ 67 mg/24 h respectively), uricosuria (417 ñ 171 and 431 ñ 121 mg/24 h respectively) or urinary magnesium (55 ñ 19 and 62 ñ 21 mg/24 h respectively, whereas urinary citrate was lower (219 ñ 172 vs 319 ñ 179 mg/24 h in controls p <0.05). All patients had a normal renal functions, urinary acidification and intravenous pyelogram. Seven percent of patients with nephrolithiasis had hypercalciuria, 2.4 percent had hyperuricosuria, 68.3 percent had a low urinary citrate and 44.4 percent had low urinary magnesium. Conclusions: in this sample, there is a strong association of nephrolithiasis with low levels of crystallization inhibitors in special with urinary citrate, a crystallization inhibitor

Serum osteocalcin in normal menstrual cycle

La concentración de Osteocalcina (OC) es un buen marcador del remodelamiento óseo. La influencia del estradiol sobre la OC ha sido descrita en el embarazo, lactancia y postmenopausia. No existe acuerdo si los cambios en la concentración plasmática de estradiol del ciclo menstrual normal se relacionan con modificaciones de la OC. En este estudio la OC sérica fue medida seriadamente a lo largo de un ciclo mesntrual normal en 4 voluntarias sanas, libres de toda medicación (edad 33-38 años). Durante el estudio las mujeres mantuvieron su dieta y actividad física habitual. A partir del día 4 ó 5 del ciclo, se tomaron muestras de sangre entre las 09 y 11 am, cada 2 ó 3 días, para determinación de OC, FSH, LH, E2 y P. Se realizó seguimiento folicular ecográfico día por medio entre los días 7 y 18 del ciclo. Las determinaciones hormonales fueron realizadas por RIA utilizando reactivos aportados por la OMS. La OC fue determinada por RIA (incstar OC 125 I Kit, Incstar, Stillwater, MN) con límite de sensibilidad de 0,78 ng/ml y coeficiente de variación intraensayo de 4,6%. Todas las muestras fueron corridas en el mismo ensayo. El análisis estadístico incluyó la prueba de Kolmogorow-Smirnow (KS) y el coeficiente de correlación temporal de Sperman (S). Las concentraciones sanguíneas de FSH, LH, E2 y P cambiaron durante el estudio de acuerdo al caráter ovulatorio del ciclo. Como se muestra en la Tabla 1, las concentraciones de OC permanecieron estables a lo largo del ciclo en cada una de las 4 mujeres. Los valores de OC se expresan como promedio ñ ES de todas las muestras tomadas a lo largo del ciclo de cada mujer. d*: diferencia absoluta máxima entre el valor observado y la distribución teórica normal según la prueba de Kolmogorov. r**:, coeficiente de correlación temporal para los valores de OC. En conclusión, aparece como independiente de las variaciones de las gonadotrofinas y esteroides ováricos del ciclo menstrual normal. De este modo, la evaluación de la formación ósea no debería ser influenciada por el día o la fase del ciclo menstrual en el cual la muestra es obtenida

Serum osteocalcin in normal menstrual cycle

La concentración de Osteocalcina (OC) es un buen marcador del remodelamiento óseo. La influencia del estradiol sobre la OC ha sido descrita en el embarazo, lactancia y postmenopausia. No existe acuerdo si los cambios en la concentración plasmática de estradiol del ciclo menstrual normal se relacionan con modificaciones de la OC. En este estudio la OC sérica fue medida seriadamente a lo largo de un ciclo mesntrual normal en 4 voluntarias sanas, libres de toda medicación (edad 33-38 años). Durante el estudio las mujeres mantuvieron su dieta y actividad física habitual. A partir del día 4 ó 5 del ciclo, se tomaron muestras de sangre entre las 09 y 11 am, cada 2 ó 3 días, para determinación de OC, FSH, LH, E2 y P. Se realizó seguimiento folicular ecográfico día por medio entre los días 7 y 18 del ciclo. Las determinaciones hormonales fueron realizadas por RIA utilizando reactivos aportados por la OMS. La OC fue determinada por RIA (incstar OC 125 I Kit, Incstar, Stillwater, MN) con límite de sensibilidad de 0,78 ng/ml y coeficiente de variación intraensayo de 4,6%. Todas las muestras fueron corridas en el mismo ensayo. El análisis estadístico incluyó la prueba de Kolmogorow-Smirnow (KS) y el coeficiente de correlación temporal de Sperman (S). Las concentraciones sanguíneas de FSH, LH, E2 y P cambiaron durante el estudio de acuerdo al caráter ovulatorio del ciclo. Como se muestra en la Tabla 1, las concentraciones de OC permanecieron estables a lo largo del ciclo en cada una de las 4 mujeres. Los valores de OC se expresan como promedio ñ ES de todas las muestras tomadas a lo largo del ciclo de cada mujer. d*: diferencia absoluta máxima entre el valor observado y la distribución teórica normal según la prueba de Kolmogorov. r**:, coeficiente de correlación temporal para los valores de OC. En conclusión, aparece como independiente de las variaciones de las gonadotrofinas y esteroides ováricos del ciclo menstrual normal. De este modo, la evaluación de la formación ósea no debería ser influenciada por el día o la fase del ciclo menstrual en el cual la muestra es obtenida (AU)