Protein Metabolism in Turner Syndrome and the Impact of Hormone Replacement Therapy

Claus Hjbjerg Gravholt, Anne Lene Riis, Niels Mller, Jens Sandahl Christiansen Disclosures Clin Endocrinol. 2007;67(3):413-418. Background: Studies have documented an altered body composition in Turner syndrome (TS). Body fat is increased and muscle mass is decreased. Ovarian failure necessitates substitution with female hormone replacement therapy (HRT), and HRT induces favourable changes in body composition. It is unknown how HRT affects protein metabolism. Aim: To test whether alterations in body composition before and after HRT in TS are a result of altered protein metabolism. Design: We performed a randomized crossover study with active treatment (HRT in TS and oral contraceptives in controls) or no treatment. Materials and Methods: We studied eight women (age 297 56 (mean SD) years) with TS, verified by karyotype, and eight age-matched controls (age 273 49 years). All subjects underwent a 3-h study in the postabsorptive state. Protein dynamics of the whole body and of the forearm muscles were measured by an amino acid tracer dilution technique using [ 15N]phenylalanine and [ 2H 4]tyrosine. Substrate metabolism was examined by indirect calorimetry. Results: Energy expenditure was comparable among TS and controls, and did not change during active treatment. Whole-body phenylalanine and tyrosine fluxes were similar in the untreated situations, and did not change during active treatment. Amino acid degradation and protein synthesis were similar in all situations. Muscle protein breakdown was similar among groups, and was not affected by treatment. Muscle protein synthesis rate and forearm blood flow did not differ among groups or due to treatment. Conclusion: Protein metabolism in TS is comparable to controls, and is not affected by HRT. Turner syndrome (TS) is characterized by the absence of a part of or the entire X chromosome, and the typical stigmata are short stature, primary amenorrhoea, oestrogen insufficiency and cardiovascular malformations. Body composition is distinctly altered, with a reduced muscle mass and increased fat mass, often despite a body mass index (BMI) within the normal range. [1-3] Body composition can be modified by GH treatment, habitually given for increasing height during childhood and adolescence in TS. [4,5] Studies in adults have documented excess visceral and hepatic fat deposition. [6,7] Morbidity and mortality are increased in TS due to risk of osteoporosis and fractures, type 2 diabetes, congenital and ischaemic heart disease, hypertension, and stroke. [8-10] More generally, mortality is increased in premature ovarian failure (POF) and female patients with hypopituitarism lacking oestrogen, [11-14] and it has been speculated, but not substantiated, that oestrogen deficiency is the major causative factor. As most women with TS are devoid of oestradiol (and progesterone) production from the ovaries, the standard therapy is sex hormone replacement therapy (HRT), starting with induction of puberty for a couple of years,

and then as a combination therapy with a gestagen for some 3040 years until the normal age of menopause when treatment can be terminated. Oestradiol is an anabolic hormone, but a study in TS children failed to show any effect on whole-body protein metabolism. [15] In pubertal rats, oestradiol inhibits fat and promotes muscle accumulation. [16] In women just after the advent of menopause, a reduction in muscle power occurs, [17] which can be counteracted by treatment with female sex steroids. [18] Treatment with female sex hormones to previously untreated postmenopausal women [19,20] or women with TS increases muscle mass and reduces fat mass. [21] Thus, we hypothesized that in adults with TS, the decreased lean body and muscle mass could relate to increased overall protein loss and that HRT could reverse this. As muscle is the major protein reservoir, we specifically assessed amino acid turnover in forearm muscle. Energy expenditure metabolism was examined by indirect calorimetry.

We studied eight women with TS (age 297 56 (mean SD); range 2438 years), verified by karyotype, and eight age-matched controls (age 273 49; range 2436 years) ( Table 1 ). The study was a randomized crossover study, with durations of 2 months, each completed by 2 study days (women were treated for two full menstrual cycles and were studied during the beginning of the third cycle). A 2-month wash-out period was introduced before each study period where the usual treatment of HRT in TS or contraceptive pill therapy in controls was stopped. The treatment regimen was given in random order to the individual participants. All women with TS were nonmenstruating and had required HRT for several years in order to menstruate regularly. Thyroid hormone levels were normal in all study individuals. Women with TS were treated with oral hormone substitution consisting of 2 mg 17-oestradiol/day for days 112, 2 mg 17-oestradiol/day plus 1 mg norethisterone acetate/day for days 1322 and 1 mg 17-oestradiol/day for days 2328 (Trisekvens, Novo Nordisk, Denmark), or no treatment for 2 months. Control subjects were treated with oral contraceptives (OCT) consisting of 75 g ethinyloestradiol and 30 g gestoden (Minulet, Wyeth, Denmark), or no treatment for 2 months. All subjects were studied in the early follicular stage (days 510) of the menstrual cycle or during the corresponding phase of the HRT (equivalent to days 510 of the HRT period where TS subjects receive 2 mg 17-oestradiol/day per contraceptive cycle). None of the participants were smokers or received medication other than HRT/contraceptives. All subjects received oral and written information concerning the study prior to giving written informed consent. The protocol was approved by the Aarhus County Ethical Scientific Committee (no. 1996/3561). Analysis of beat-to-beat variation and 24-h ambulatory blood pressure measurements from this study have been presented previously. [22] We used L-[ 15N]phenylalanine, L-[ 15N]tyrosine and L-[ 2H 4]tyrosine from Cambridge Isotope Laboratories (Andover, MA). The chemical, isotopic and optical purity of the isotopes was tested before use. Solutions were prepared under sterile conditions and were shown to be free of bacteria and pyrogens before use. The participants were admitted to the Clinical Research Centre in the morning on the day of the examinations. The investigations were carried out in the postabsorptive state the morning after an overnight fast (1012 h) without any caffeine consumption; only ingestion of tap water was allowed, and the participants were placed in the supine position under thermo-

neutral conditions. After an initial bed-rest of at least 45 min, indirect calorimetry (Deltatrac Metabolic Monitor, Datex, Helsinki, Finland) with a ventilated hood at 40 l/min was performed; energy expenditure (EE) and respiratory quotient (RQ) were measured. Resistance and impedance were measured and fat mass (FM), fat free mass (FFM) and total body water (TBW) were determined by bioeletrical impedance using BIA 101/S (RJL Systems, Detroit, MI); TBW, lean body mass and FM were calculated as a percentage of body weight. [23] BMI was calculated as weight (kg) divided by height (m) squared, and the waist-to-hip (W/H) ratio was determined in the supine position. One intravenous catheter (Viggo, Helsingborg, Sweden) was placed in an antecubital vein for infusions, another in the contralateral antecubital vein for deep venous samples, and a third in a superficial vein draining the ipsilateral hand, which was heated in a box with an air temperature of 65C to provide arterialized blood. [24] Preceding every deep venous sampling, forearm blood flow was determined by venous occlusion plethysmography. After priming the amino acid pool with bolus injections of [ 15N]phenylalanine (07 mg/kg), [ 15N]tyrosine (03 mg/kg) and [ 2H 4]tyrosine (05 mg/kg), continuous infusions of [ 15N]phenylalanine (07 mg/kg/h) and [ 2H 4]tyrosine (05 mg/kg/h) were maintained for 3 h. After 150 min of continuous infusion, steady state is accomplished, and blood samples were taken in triplicate during the last 30 min of each study. Calculations of phenylalanine kinetics. The equations of Thompson et al. [25] were used for measurements of whole-body phenylalanine kinetics. Phenylalanine flux ( Q Phe) and tyrosine flux ( Q Tyr) were calculated as follows: Q = i [( E i/ E p) 1] where i is the rate of tracer infusion (mol/kg/h) and E i and E p are enrichment of the tracer infused and plasma enrichment of the tracer at the isotopic plateau, respectively. The rate of phenylalanine conversion by hydroxylation to tyrosine ( I pt) was calculated as follows: I pt = Q Tyr ([ 15N]Tyr ei/[ 15N]Phe ei) [ Q Phe/( I Phe + Q Phe)] where [ 15N]Tyr ei and [ 15N]Phe ei are the isotopic enrichments of the respective tracers in plasma and I Phe is the infusion rate of [ 15N]phenylalanine (mol/kg/h). Phenylalanine incorporation into protein is calculated by subtracting I pt from Q Phe because phenylalanine is irreversibly lost from the bloodstream either by its hydroxylation into tyrosine or by incorporation into protein. In the forearm study, phenylalanine balance (PheBal) was calculated using Fick's principle: PheBal = (Phe a Phe v) F where Phe a and Phe v are arterial and deep venous phenylalanine concentrations, respectively, and F is the blood flow in the forearm. Regional phenylalanine kinetics were calculated using the equations described by Nair et al. [26] The forearm protein breakdown represented by phenylalanine rate of appearance ( R a Phe) was calculated as follows: [27] R a Phe = Phe a [(Phe Ea/Phe Ev) 1] F

where Phe Ea and Phe Ev represent phenylalanine isotopic enrichment in arteries and veins, respectively. The local rate of disappearance, which represents the muscle protein synthesis rate, was calculated as: R d Phe = PheBal + R a Phe where R d Phe is the forearm protein synthesis rate. Enrichment of [ 15N]phenylalanine, [ 15N]tyrosine and [ 2H 4]tyrosine was measured by mass spectrometry as their t-butyldimethylsilyl ether derivates under electron ionization conditions, and concentrations of phenylalanine and tyrosine were measured using L-[ 2H 13 [26] Glucose was measured by the 8]phenylalanine and L-[ C 6]tyrosine as internal standards. glucose oxidase method. We used a two-site enzyme-linked immunosorbent assay (ELISA) to measure serum insulin. A double monoclonal immunofluorometric assay (Delfia, Wallac Oy, Turku, Finland) was used to measure serum GH while plasma glucagon was measured with an in-house radioimmunoassay (RIA). [28] Serum IGF-I was measured with an in-house time-resolved fluoroimmunoassay (TR-FIA). Serum free fatty acids (FFA) were determined by a colorimetric method using a commercial kit (Wako Chemicals, Neuss, Germany). Serum SHBG was measured by a commercial TR-FIA (Delfia). Serum oestradiol, testosterone, FSH and LH were measured by commercial TR-FIAs (Delfia) with detection limits of 005 nmol/l, 03 nmol/l, 006 and 0.05 IU/l, respectively. Intra- and interassay coefficients of variation were both below 8% in the FSH and LH assays. All statistical calculations were performed with SPSS for Windows version 130 (SPSS Inc., Chicago, IL). Data were checked for parametric distribution. Data were examined by Student's two-tailed paired and unpaired t-tests when appropriate. Results are expressed as mean standard deviation (SD). Significance levels less than 5% were considered significant. TS women were shorter and lighter than untreated controls, but with a greater BMI, although this difference did not reach statistical significance ( Table 2 ). Similarly, FM tended to be higher in TS, while FFM and TBW tended to be lower in TS. Weight, BMI, FM, FFM and TBW did not change during the study period in both TS and controls ( Table 2 ), although there was a tendency for an increase in FFM and a corresponding decrease in FM in TS during treatment. Energy expenditure and RQ were comparable among TS and controls and did not change during active treatment ( Table 2 ). Serum oestradiol, SHBG, FSH and LH levels were in the postmenopausal range before treatment in untreated TS, as expected, and were significantly influenced by HRT. Serum oestradiol and SHBG normalized while FSH and LH were still elevated, as is normally seen when 2 mg of 17-oestradiol is given. Serum testosterone was slightly lower in TS and did not change during HRT. Serum oestradiol, SHBG and testosterone changed significantly in controls during OCT, as expected, and FSH and LH declined but not significantly ( Table 2 ). The changes indicated an overall increased oestrogenization in controls during contraceptive treatment. Circulating levels of serum GH and IGF-I were comparable among groups. There was no change in glucose and insulin due to treatment among TS or controls (results not shown), but plasma glucose was slightly lower in controls while serum insulin and homeostatic model assessment (HOMA) index were similar among TS and controls. Serum

FFA was identical in TS and controls ( Table 1 ) and did not change due to treatment (results not shown). None of the participants reported any side-effects to treatment. Isotopic enrichments reached a plateau at the end of the study period and were similar in all study periods (data not shown). Whole-body phenylalanine and tyrosine fluxes were similar in the untreated situations and did not change during active treatment in TS ( Table 3 ) or controls ( P-values not shown). Phenylalanine conversion to tyrosine (reflecting amino acid degradation) and protein synthesis (phenylalanine disposal not accounted for by phenylalanine conversion to tyrosine) was similar in all situations. During the forearm study we saw a net release of phenylalanine in all study groups. Muscle protein breakdown assessed by phenylalanine rate of appearance was similar among groups and was not affected by treatment. Muscle protein synthesis rate and forearm blood flow did not differ among groups or due to treatment. The present study was designed to define amino acid and protein metabolism in muscle and at the whole-body level in patients with TS and to test the influence of HRT on protein metabolism in a model of female hypogonadism, that is TS, and to compare protein metabolism with an age-matched control group during a normal oestrogenized state, as well as during a hyper-oestrogenized state (OCT). We did not detect any impact of HRT on protein metabolism during 2 months, nor did we find any difference in protein metabolism in comparison with a control group of healthy women. We studied TS women in the hypogonadal state with very low levels of 17-oestradiol and reciprocal elevation of FSH and LH; [29] during treatment we achieved an appropriate level of oestrogenization in comparison with controls, although FSH and LH were not completely normalized in all TS. Despite this, protein metabolism was not affected and we did not detect any change in energy expenditure. These results are consonant with findings in pubertal girls with TS, in whom whole-body, but not regional, protein metabolism was studied with a leucine tracer and found to be normal. [15] By contrast, oestrogen has profound anabolic effects on calcium metabolism in terms of increased calcium absorption, retention, and diminished whole-body calcium turnover during active treatment. [30] However, in the study by Mauras, [15] the lack of effect on protein metabolism might have been due to the fact that the patients studied were prepubertal and they were treated with low-dose oestradiol (most were given synthetic ethinyl oestradiol) for pubertal induction purposes, whereas we used a standard dose of 2 mg of 17-oestradiol usually given to adults. The HRT regimen chosen in this study is the standard therapy in most European countries given to women with TS or POF, consisting of natural human 17-oestradiol and a gestagen, and not conjugated equine oestrogens, which is commonly used in the USA and known to be a composite of a number of oestrogenic compounds. Oestradiol is a weak anabolic hormone that affects body composition with increases in lean body mass and decreases in fat mass. A reduction of muscle power is seen after menopause, [17] but this reduction can be avoided by substitution with female sex steroids. [18] Treatment with HRT for months in previously untreated postmenopausal women [19] or women with TS increases muscle mass and reduces fat mass. [21] In postmenopausal women receiving HRT, fat accumulation is prevented and lipoprotein lipase activity and lipolysis increases ( in vitro), again resembling the pattern seen in premenopausal women, [19,31,32] in whom the lipolytic sensitivity and responsiveness ( in vitro) are higher. [33] One study, using a whole-body tracer dilution technique, found fat metabolism to be higher during treatment with oestradiol than without. There was no

difference in catecholamine-stimulated lipolysis, [34] although another study suggested reduced lipolysis during treatment with oestradiol because the number of -adrenergic receptors was increased whereas the number of -adrenergic receptors was down-regulated in postmenopausal women. [35] Taken together, studies of different populations and of differing tissues and regions thus suggest that oestrogen might affect protein metabolism, and it may be that we simply missed the time window in which HRT significantly affects protein metabolism. A new steady state may have ensued after 2 months of HRT, and studying protein metabolism after a shorter course of HRT might have revealed significant changes. The fact that a changed body composition is seen in hypo-oestrogenic women lends credit to this notion. Prolonging the study period to more than 2 months, however, would probably not have yielded differences in protein metabolism because a new steady state would probably arise. Another possibility is that changes in protein metabolism are very subtle and therefore a larger number of study subjects would be required. The sample size in the present study was small and this might have affected our making firm conclusions, although we did not detect even a trend towards an effect of HRT (or the contraceptive pill) on protein metabolism. The dose of oestradiol (2 mg) may well have been insufficient, inasmuch as we did not see a complete normalization of FSH (and LH), and it may be that a doubling of the dose to 4 mg of oestradiol would have affected protein metabolism significantly. Currently, new studies indicate that the traditional dose of 2 mg of oestradiol used in TS and other conditions of POF may be too low in terms of normalizing the cardiovascular system and for normal growth of the uterus. [36,37] We chose to use the contraceptive pill in controls and not HRT as in the TS subjects, because HRT given to normally cycling women can induce irregular bleeding and does not work as a contraceptive because of irregular suppression of FSH. In the controls, who moved from a eu-oestrogenized to a hyper-oestrogenized state during OCT, we also did not see any changes in protein metabolism or measures of body composition. Apart from oestradiol, most circulating hormones were comparable in TS and controls, although serum testosterone was slightly diminished, as found previously, [38] and it is not likely that differences in insulin or other hormones have affected our results. Furthermore, because oestradiol only affects insulin sensitivity marginally, [39] if at all, it is not likely that HRT works through changes in insulin levels or through changes in insulin sensitivity. In conclusion, we did not detect any effect of 2 months of HRT in TS on protein metabolism. Treatment with a contraceptive pill for 2 months in normal controls also did not affect protein metabolism, and thus protein metabolism is similar during hypo-, eu- and hyperoestrogenized states.

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35. Pedersen, S.B., Kristensen, K., Hermann, P.A., Katzenellenbogen, J.A. & Richelsen, B. (2004) Estrogen controls lipolysis by up-regulating alpha2A-adrenergic receptors directly in human adipose tissue through the estrogen receptor alpha. Implications for the female fat distribution. Journal of Clinical Endocrinology and Metabolism, 89, 18691878 36. Snajderova, M., Mardesic, T., Lebl, J., Gerzova, H., Teslik, L. & Zapletalova, J. (2003) The uterine length in women with Turner syndrome reflects the postmenarcheal daily estrogen dose. Hormone Research, 60, 198204 37. Ostberg, J.E., Storry, C., Donald, A.E., Attar, M.J., Halcox, J.P. & Conway, G.S. (2007) A doseresponse study of hormone replacement in young hypogonadal women: effects on intima media thickness and metabolism. Clinical Endocrinology, 66, 557564 38. Gravholt, C.H., Svenstrup, B., Bennett, P. & Christiansen, J.S. (1999) Low androgen levels in adult Turner syndrome: influence of female sex hormones and growth hormone status. Clinical Endocrinology, 50, 791800 39. Godsland, I.F. (2005) Oestrogens and insulin secretion. Diabetologia, 48, 22132220
41. Protein Metabolisme dalam Sindrom Turner dan Dampak Terapi Hormon Replacement Claus Hjbjerg Gravholt, Anne Lene Riis, Niels Moller, Jens Sandahl Christiansen Pengungkapan Clin Endocrinol. 2007; 67 (3) :413-418. Latar Belakang: Studi telah mendokumentasikan sebuah komposisi tubuh berubah di Turner syndrome (TS). Lemak tubuh meningkat dan massa otot berkurang. Kegagalan ovarium membutuhkan substitusi dengan terapi penggantian hormon wanita (HRT), dan HRT menyebabkan perubahan yang menguntungkan dalam komposisi tubuh. Tidak diketahui berapa HRT mempengaruhi metabolisme protein. Tujuan: Untuk menguji apakah perubahan dalam komposisi tubuh sebelum dan sesudah HRT di TS adalah hasil dari metabolisme protein diubah. Desain: Kami melakukan studi crossover acak dengan pengobatan aktif (HRT di TS dan kontrasepsi oral dalam kontrol) atau tanpa pengobatan. Bahan dan Metode: Kami mempelajari delapan perempuan (usia 29 7 5 (mean SD) tahun 6) dengan TS, diverifikasi oleh kariotipe, dan delapan usia-cocok kontrol (usia 27 3 4 9 tahun). Semua orang menjalani studi 3-jam dalam keadaan postabsortif. Protein dinamika seluruh tubuh dan otot-otot lengan diukur dengan teknik perunut asam amino pengenceran menggunakan [15N] fenilalanin dan [2H 4] tirosin. Metabolisme Substrat diperiksa oleh kalorimetri langsung. Hasil: Pengeluaran energi adalah sebanding antara TS dan kontrol, dan tidak berubah selama pengobatan aktif. Fluks seluruh tubuh fenilalanin dan tirosin adalah serupa dalam situasi yang tidak diobati, dan tidak berubah selama pengobatan aktif. Asam amino degradasi dan sintesis protein adalah serupa dalam segala situasi. Kerusakan otot protein adalah serupa di antara kelompok-kelompok, dan tidak terpengaruh oleh pengobatan. Otot sintesis protein rate dan aliran darah lengan tidak berbeda antara kelompok-kelompok atau akibat pengobatan. Kesimpulan: Protein metabolisme dalam TS sebanding dengan kontrol, dan tidak terpengaruh oleh HRT. Sindrom Turner (TS) ditandai dengan tidak adanya sebagian atau seluruh kromosom X, dan stigmata khas adalah perawakan pendek, primer amenorea, estrogen insufisiensi dan malformasi kardiovaskular. Komposisi tubuh yang jelas diubah, dengan massa otot

berkurang dan massa lemak meningkat, seringkali meskipun indeks massa tubuh (BMI) dalam kisaran normal. [1-3] Komposisi tubuh dapat dimodifikasi dengan perlakuan GH, biasanya diberikan untuk tinggi meningkat selama masa kanak-kanak dan remaja di TS. [4,5] Studi pada orang dewasa telah mendokumentasikan penumpukan lemak visceral kelebihan dan hati. [6,7] Morbiditas dan kematian yang meningkat pada TS karena risiko osteoporosis dan patah tulang, diabetes tipe 2, penyakit jantung bawaan dan iskemik, hipertensi, dan stroke. [8-10] Secara umum, angka kematian meningkat pada kegagalan ovarium prematur (POF) dan pasien wanita dengan hypopituitarism kurang estrogen, [11-14] dan telah berspekulasi, tetapi tidak dibuktikan, bahwa kekurangan estrogen adalah faktor penyebab utama. Seperti sebagian besar wanita dengan TS adalah tanpa estradiol (dan progesteron) produksi dari indung telur, terapi standar seks terapi penggantian hormon (HRT), dimulai dengan induksi pubertas selama beberapa tahun, dan kemudian sebagai terapi kombinasi dengan gestagen untuk beberapa tahun sampai usia 30-40 normal menopause ketika pengobatan dapat dihentikan. Estradiol adalah hormon anabolik, tetapi studi pada anak-anak TS gagal menunjukkan efek pada seluruh tubuh metabolisme protein. [15] Pada tikus pubertas, estradiol menghambat akumulasi lemak dan mempromosikan otot. [16] Pada wanita setelah munculnya menopause, penurunan kekuatan otot terjadi, [17] yang dapat dinetralkan oleh pengobatan dengan steroid seks perempuan. [18] Pengobatan dengan hormon seks perempuan untuk perempuan postmenopause yang sebelumnya tidak diobati [19,20] atau wanita dengan TS meningkatkan massa otot dan mengurangi massa lemak. [21] Dengan demikian, kita hipotesis bahwa pada orang dewasa dengan TS, tubuh ramping menurun dan massa otot bisa berhubungan dengan hilangnya protein meningkat secara keseluruhan dan HRT yang bisa membalikkan ini. Karena otot adalah protein utama waduk, kami secara khusus menilai omset asam amino pada otot lengan. Metabolisme energi pengeluaran diperiksa oleh kalorimetri langsung. Kami mempelajari delapan wanita dengan TS (umur 29 7 5 6 (mean SD), kisaran 24-38 tahun), diverifikasi oleh kariotipe, dan delapan usia-cocok kontrol (usia 27 3 4 9; kisaran 24 -36 tahun) (Tabel 1). Penelitian ini merupakan studi crossover acak, dengan jangka waktu dari 2 bulan, masing-masing diisi oleh 2 hari studi (wanita dirawat selama dua siklus menstruasi penuh dan dipelajari selama awal siklus ketiga). A 2-bulan mencuci-out periode diperkenalkan sebelum setiap periode studi di mana pengobatan biasa HRT di TS atau terapi pil kontrasepsi dalam kontrol dihentikan. Rejimen pengobatan diberikan secara acak ke peserta individu. Semua wanita dengan TS yang nonmenstruating dan telah diperlukan HRT selama beberapa tahun untuk menstruasi teratur. Kadar hormon tiroid yang normal dalam semua individu studi. Wanita dengan TS diobati dengan substitusi hormon oral yang terdiri dari 2 17-oestradiol/day mg selama 1-12 hari, 2 mg 17-oestradiol/day ditambah 1 mg norethisterone asetat / hari untuk hari 13-22 dan 1 mg 17-estradiol / hari untuk hari 23-28 (Trisekvens , Novo Nordisk, Denmark), atau ada pengobatan selama 2 bulan. Subyek kontrol diobati dengan kontrasepsi oral (OCT) yang terdiri dari 75 ug etinilestradiol dan 30 ug gestoden (Minulet, Wyeth, Denmark), atau ada pengobatan selama 2 bulan. Semua subjek yang dipelajari dalam tahap folikuler awal (5-10 hari) dari siklus menstruasi atau selama fase sesuai HRT (setara dengan 5-10 hari dari periode HRT mana subyek TS menerima 2 mg per 17-oestradiol/day kontrasepsi siklus). Tak satu pun dari peserta adalah perokok atau

menerima obat selain HRT / kontrasepsi. Semua subjek menerima informasi lisan dan tertulis mengenai studi sebelum memberikan persetujuan tertulis. Protokol ini disetujui oleh Komite Etis di County Aarhus Ilmiah (no. 1996/3561). Analisis beat-to-beat variasi dan 24-jam pengukuran tekanan darah rawat jalan dari penelitian ini telah dipresentasikan sebelumnya. [22] Kami menggunakan L-[15N] fenilalanin, L-[15N] tirosin dan L-[2H 4] tirosin dari Cambridge Isotop Laboratories (Andover, MA). Bahan kimia, isotop dan kemurnian optik dari isotop diuji sebelum digunakan. Solusi disusun berdasarkan kondisi steril dan terbukti bebas dari bakteri dan pirogen sebelum digunakan. Para peserta dirawat di Pusat Penelitian Klinis di pagi hari pada hari pemeriksaan. Penyelidikan dilakukan di negara postabsortif pagi hari setelah puasa semalaman (10-12 jam) tanpa konsumsi kafein, konsumsi hanya air keran diizinkan, dan peserta ditempatkan dalam posisi terlentang di bawah termo-netral kondisi. Setelah awal tidur-sisa setidaknya 45 menit, kalorimetri langsung (Deltatrac Metabolik Monitor, Datex, Helsinki, Finlandia) dengan hood ventilasi pada 40 l / min dilakukan, pengeluaran energi (EE) dan kecerdasan pernapasan (RQ) diukur . Perlawanan dan impedansi diukur dan massa lemak (FM), massa bebas lemak (FFM) dan air tubuh total (TBW) ditentukan oleh impedansi bioeletrical menggunakan BIA 101 / S (RJL Systems, Detroit, MI), TBW, massa tubuh tanpa lemak dan FM dihitung sebagai persentase dari berat badan. [23] IMT dihitung sebagai berat badan (kg) dibagi tinggi badan (m) kuadrat, dan pinggang-panggul (W / H) rasio ditentukan dalam posisi terlentang. Satu kateter intravena (Viggo, Helsingborg, Swedia) ditempatkan dalam vena antecubital untuk infus, yang lain dalam vena antecubital kontralateral untuk sampel vena dalam, dan ketiga dalam vena superfisial menguras sisi ipsilateral, yang dipanaskan dalam kotak dengan suhu udara dari 65 C untuk menyediakan darah arterialized. [24] Mendahului setiap sampel vena dalam, aliran darah lengan ditentukan oleh vena oklusi plethysmography. Setelah priming kolam asam amino dengan suntikan bolus *15N+ fenilalanin (0 7 mg / kg), *15N+ tirosin (0 3 mg / kg) dan *2H 4+ tirosin (0 5 mg / kg), infus terus menerus *15N+ fenilalanin (0 7 mg / kg / jam) dan *2H 4+ tirosin (0 5 mg / kg / h) dipertahankan selama 3 jam. Setelah 150 menit dari infus kontinu, steady state dicapai, dan sampel darah diambil dalam rangkap tiga selama 30 menit terakhir dari studi masing-masing. Perhitungan kinetika fenilalanin. Persamaan dari Thompson et al. [25] yang digunakan untuk pengukuran seluruh tubuh kinetika fenilalanin. Fenilalanin fluks (Q Phe) dan fluks tirosin (Tyr Q) dihitung sebagai berikut: Q = i [(E i / E p) - 1] dimana i adalah tingkat tracer infus (mol / kg / jam) dan E i dan E p adalah pengayaan dari tracer diinfus dan pengayaan plasma pelacak di dataran tinggi isotop, masing-masing. Tingkat konversi fenilalanin oleh hidroksilasi tirosin untuk (I pt) dihitung sebagai berikut: Saya pt = Q Tyr ([15N] Tyr ei / [15N] Phe ei) [Q Phe / (I + Q Phe Phe)] mana [15N] Tyr ei dan [15N] Phe ei adalah pengkayaan isotop dari pelacak masing dalam plasma dan saya Phe adalah tingkat infus [15N] fenilalanin (mol / kg / h). Penggabungan fenilalanin menjadi protein dihitung dengan mengurangkan Saya pt dari Q Phe karena fenilalanin ireversibel hilang dari aliran darah baik oleh hidroksilasi tirosin ke dalam atau dimasukkan ke dalam protein. Dalam studi lengan, keseimbangan fenilalanin (PheBal) dihitung menggunakan prinsip Fick:

PheBal = (a Phe - Phe v) F mana Phe Phe dan v adalah arteri dan vena dalam konsentrasi fenilalanin, masing-masing, dan F adalah aliran darah di lengan bawah. Daerah fenilalanin kinetika dihitung dengan menggunakan persamaan dijelaskan oleh Nair et al. [26] Protein lengan breakdown diwakili oleh tingkat fenilalanin penampilan (R Phe) dihitung sebagai berikut: [27] R a = Phe Phe a [(Phe Ea / Phe Ev) - 1] F mana Phe Phe Ea dan Ev merupakan pengayaan isotop fenilalanin dalam arteri dan vena, masing-masing. Tingkat lokal penghilangan, yang merupakan tingkat otot sintesis protein, dihitung sebagai: R d = Phe PheBal + R a Phe di mana R d Phe adalah protein lengan tingkat sintesis. Pengayaan [15N] fenilalanin, [15N] tirosin dan [2H 4] tirosin diukur dengan spektrometri massa sebagai t-butyldimethylsilyl mereka turunan eter dalam kondisi ionisasi elektron, dan konsentrasi fenilalanin dan tirosin yang diukur dengan menggunakan L-[2H 8] fenilalanin dan L-[13C 6] tirosin sebagai standar internal. [26] Glukosa diukur dengan metode oksidase glukosa. Kami menggunakan assay enzim-linked immunosorbent dua-situs (ELISA) untuk mengukur insulin serum. Sebuah uji immunofluorometric ganda monoklonal (Delfia, Wallac Oy, Turku, Finlandia) digunakan untuk mengukur serum GH sementara plasma glukagon diukur dengan radioimmunoassay in-house (RIA). [28] Serum IGF-I diukur dengan fluoroimmunoassay di rumah waktu diselesaikan (TR-FIA). Serum asam lemak bebas (FFA) ditentukan dengan metode kolorimetri menggunakan kit komersial (Wako Kimia, Neuss, Jerman). Serum SHBG diukur dengan komersial TR-FIA (Delfia). Serum estradiol, testosteron, FSH dan LH diukur dengan komersial TR-FIAS (Delfia) dengan batas deteksi 0 05 nmol / l, 0 3 nmol / l, 0 06 dan 0,05 IU / l, masing-masing. Koefisien intra dan interassay variasi berdua di bawah 8% pada FSH dan LH tes. Semua perhitungan statistik dilakukan dengan SPSS for Windows versi 13 0 (SPSS Inc, Chicago, IL). Data diperiksa untuk distribusi parametrik. Data diperiksa oleh dua-ekor Mahasiswa dipasangkan dan berpasangan t-tes saat yang tepat. Hasil dinyatakan sebagai mean standar deviasi (SD). Tingkat signifikansi kurang dari 5% dianggap signifikan. TS wanita yang lebih pendek dan lebih ringan dari kontrol tidak diobati, namun dengan BMI yang lebih besar, meskipun perbedaan ini tidak bermakna secara statistik (Tabel 2). Demikian pula, FM cenderung lebih tinggi di TS, sedangkan FFM dan TBW cenderung lebih rendah di TS. Berat, BMI, FM, dan FFM TBW tidak berubah selama masa studi di kedua TS dan kontrol (Tabel 2), meskipun ada kecenderungan peningkatan FFM dan penurunan nilai FM di TS selama pengobatan. Pengeluaran energi dan RQ adalah sebanding antara TS dan kontrol dan tidak berubah selama pengobatan aktif (Tabel 2). Serum estradiol, SHBG, FSH dan LH berada di kisaran pascamenopause sebelum pengobatan di TS tidak diobati, seperti yang diharapkan, dan secara signifikan dipengaruhi oleh HRT. Serum estradiol dan SHBG normal sementara FSH dan LH masih tinggi, seperti yang biasanya terlihat ketika 2 mg 17-estradiol diberikan. Testosteron serum sedikit lebih rendah di TS dan tidak berubah selama HRT. Serum estradiol, SHBG dan testosteron berubah secara signifikan dalam kontrol selama Oktober, seperti yang diharapkan FSH, LH dan dan menurun tetapi tidak signifikan (Tabel 2). Perubahan mengindikasikan oestrogenization meningkat secara keseluruhan dalam kontrol selama pengobatan kontrasepsi. Tingkat sirkulasi serum

GH dan IGF-I adalah sebanding antara kelompok. Tidak ada perubahan glukosa dan insulin akibat pengobatan antara TS atau kontrol (hasil tidak ditampilkan), tetapi glukosa plasma sedikit lebih rendah di kontrol, sementara insulin serum dan homeostatic model assessment (HOMA) Indeks yang serupa di antara TS dan kontrol. Serum FFA adalah identik dalam TS dan kontrol (Tabel 1) dan tidak berubah karena pengobatan (hasil tidak ditampilkan). Tak satu pun dari peserta melaporkan efek samping pengobatan. Pengkayaan isotop mencapai dataran tinggi pada akhir masa studi dan adalah serupa dalam semua periode studi (data tidak ditampilkan). Fluks seluruh tubuh fenilalanin dan tirosin adalah serupa dalam situasi yang tidak diobati dan tidak berubah selama pengobatan aktif di TS (Tabel 3) atau kontrol (P-nilai-nilai tidak ditampilkan). Konversi fenilalanin menjadi tirosin (mencerminkan degradasi asam amino) dan sintesis protein (pembuangan fenilalanin tidak diperhitungkan oleh konversi fenilalanin menjadi tirosin) adalah serupa dalam semua situasi. Selama studi lengan kami melihat rilis bersih fenilalanin pada semua kelompok studi. Otot protein breakdown dinilai oleh tingkat fenilalanin penampilan adalah serupa di antara kelompok-kelompok dan tidak terpengaruh oleh pengobatan. Otot sintesis protein rate dan aliran darah lengan tidak berbeda antara kelompok-kelompok atau akibat pengobatan. Penelitian ini dirancang untuk menentukan asam amino dan metabolisme protein di otot dan di tingkat seluruh tubuh pada pasien dengan TS dan untuk menguji pengaruh HRT pada metabolisme protein dalam model hipogonadisme perempuan, yaitu TS, dan untuk membandingkan protein metabolisme dengan kelompok usia-cocok kontrol selama keadaan oestrogenized normal, serta selama keadaan hiper-oestrogenized (OCT). Kami tidak mendeteksi adanya dampak HRT pada metabolisme protein selama 2 bulan, atau apakah kita menemukan perbedaan dalam metabolisme protein dibandingkan dengan kelompok kontrol perempuan yang sehat. Kami mempelajari wanita TS di negara hipogonadisme dengan tingkat yang sangat rendah dari elevasi 17-estradiol dan timbal balik dari FSH dan LH, [29] selama pengobatan kami mencapai tingkat yang tepat dari oestrogenization dibandingkan dengan kontrol, meskipun FSH dan LH tidak sepenuhnya dinormalisasi dalam TS semua. Meskipun demikian, metabolisme protein tidak terpengaruh dan kami tidak mendeteksi perubahan dalam pengeluaran energi. Hasil ini sejalan dengan temuan pada anak perempuan pubertas dengan TS, di antaranya seluruh tubuh, tapi bukan regional, metabolisme protein dipelajari dengan pelacak leusin dan ditemukan menjadi normal. [15] Sebaliknya, estrogen memiliki efek anabolik mendalam pada metabolisme kalsium dalam hal penyerapan kalsium meningkat, retensi, dan seluruh tubuh berkurang omset kalsium selama pengobatan aktif. [30] Namun, dalam studi oleh Mauras, [15] kurangnya efek pada metabolisme protein mungkin karena fakta bahwa pasien yang diteliti adalah prapubertas dan mereka diperlakukan dengan dosis rendah estradiol (sebagian diberi etinil estradiol sintetik ) untuk tujuan induksi pubertas, sedangkan kami menggunakan dosis standar 2 mg 17-estradiol biasanya diberikan kepada orang dewasa. Regimen HRT yang dipilih dalam penelitian ini adalah terapi standar di negara-negara Eropa yang paling diberikan kepada perempuan dengan TS atau POF, terdiri dari estrogen alami manusia 17-estradiol dan gestagen, dan tidak kuda terkonjugasi, yang umum digunakan di Amerika Serikat dan dikenal menjadi komposit dari sejumlah senyawa estrogenik. Estradiol adalah hormon anabolik yang lemah yang mempengaruhi komposisi tubuh dengan peningkatan massa tubuh tanpa lemak dan penurunan massa lemak. Penurunan kekuatan otot terlihat setelah menopause, [17] tetapi pengurangan ini dapat

dihindari dengan substitusi dengan steroid seks perempuan. [18] Pengobatan dengan HRT selama berbulan-bulan pada wanita pascamenopause yang sebelumnya tidak diobati [19] atau wanita dengan TS meningkatkan massa otot dan mengurangi massa lemak. [21] Pada wanita menopause yang menerima HRT, akumulasi lemak dicegah dan lipoprotein lipase aktivitas dan meningkatkan lipolisis (in vitro), lagi menyerupai pola yang terlihat pada wanita premenopause, [19,31,32] di antaranya sensitivitas lipolitik dan responsif (dalam vitro) yang lebih tinggi. [33] Satu studi, menggunakan teknik-seluruh tubuh tracer pengenceran, menemukan metabolisme lemak lebih tinggi selama pengobatan dengan estradiol daripada tanpa. Tidak ada perbedaan dalam lipolisis katekolamin-dirangsang, [34] meskipun penelitian lain menyarankan lipolisis berkurang selama pengobatan dengan estradiol karena jumlah -adrenergik reseptor meningkat sedangkan jumlah -adrenergik reseptor turundiatur pada wanita postmenopause. [35] Secara keseluruhan, studi populasi yang berbeda dan berbeda dari jaringan dan daerah sehingga menunjukkan estrogen yang dapat mempengaruhi metabolisme protein, dan mungkin bahwa kita hanya merindukan jendela waktu di mana HRT secara signifikan mempengaruhi metabolisme protein. Sebuah kondisi mapan baru mungkin terjadi setelah 2 bulan HRT, dan mempelajari metabolisme protein setelah kursus singkat HRT mungkin telah mengungkapkan perubahan signifikan. Fakta bahwa komposisi tubuh berubah terlihat dalam hipo-estrogenik wanita meminjamkan kredit kepada gagasan ini. Memperpanjang masa studi lebih dari 2 bulan, bagaimanapun, mungkin tidak akan menghasilkan perbedaan dalam metabolisme protein karena kondisi mapan baru mungkin akan muncul. Kemungkinan lain adalah bahwa perubahan dalam metabolisme protein yang sangat halus dan karena itu sejumlah besar subyek penelitian akan diperlukan. Ukuran sampel dalam penelitian ini adalah kecil dan ini mungkin telah mempengaruhi kesimpulan membuat kami tegas, meskipun kami tidak mendeteksi bahkan kecenderungan efek HRT (atau pil kontrasepsi) pada metabolisme protein. Dosis estradiol (2 mg) mungkin juga telah cukup, karena kita tidak melihat normalisasi lengkap FSH (dan LH), dan mungkin bahwa dua kali lipat dari dosis ke 4 mg estradiol akan mempengaruhi metabolisme protein signifikan. Saat ini, studi baru menunjukkan bahwa dosis tradisional 2 mg estradiol digunakan dalam TS dan kondisi lain dari POF mungkin terlalu rendah dalam hal normalisasi sistem kardiovaskular dan untuk pertumbuhan normal rahim. [36,37] Kami memilih untuk menggunakan pil kontrasepsi dalam kontrol dan tidak HRT seperti dalam pelajaran TS, karena HRT diberikan kepada perempuan biasanya bersepeda dapat menginduksi perdarahan yang tidak teratur dan tidak bekerja sebagai kontrasepsi karena penindasan tidak teratur FSH. Dalam kontrol, yang pindah dari eu-oestrogenized ke keadaan hiperoestrogenized selama Oktober, kami juga tidak melihat perubahan dalam metabolisme protein atau tindakan dari komposisi tubuh. Selain estradiol, hormon beredar kebanyakan adalah sebanding pada TS dan kontrol, meskipun testosteron serum sedikit berkurang, seperti yang ditemukan sebelumnya, [38] dan tidak mungkin bahwa perbedaan insulin atau hormon lain telah mempengaruhi hasil kami. Selanjutnya, karena estradiol hanya mempengaruhi sensitivitas insulin sedikit, [39] jika sama sekali, tidak mungkin bahwa karya HRT melalui perubahan tingkat insulin atau melalui perubahan dalam sensitivitas insulin. Sebagai kesimpulan, kami tidak mendeteksi efek dari 2 bulan HRT di TS pada metabolisme protein. Pengobatan dengan pil kontrasepsi selama 2 bulan di kontrol normal juga tidak

mempengaruhi metabolisme protein, dan dengan demikian metabolisme protein serupa selama hipo-, eu-dan hiper-oestrogenized negara. 42. Undo edits 43. New! Click the words above to edit and view alternate translations. Dismiss 52. Google Translate for Business:Translator ToolkitWebsite TranslatorGlobal Market Finder