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Growth Hormone & IGF ResearchVolume 22, Issue 6, December 2012, Pages 234239

Interstitial fluid contains higher in vitro IGF bioactivity thanserum: A study utilizing the suction blister technique

Ulrick Espelunda,b, ,

Klaus Sndergaardc, d,

Peter Bjerringe,

Allan Flyvbjerga, b,

Jan Frystyka,b

Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Denmark

The Medical Research Laboratories, Department of Clinical Medicine, Aarhus

University, Aarhus C, Denmark

Department of Rheumatology, Aarhus University Hospital, Denmark

Department of Clinical Medicine, Aarhus University, Aarhus C, Denmark

Mlholm Research, Mlholm Private Hospital A/S, Vejle, Denmark

http://dx.doi.org/10.1016/j.ghir.2012.08.002,How to Cite or Link Using DOI

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Abstract

Context

Circulating insulin-like growth factors (IGFs) are bound in complexes which affect their tissue-

accessibility. Interstitial fluid is in close proximity to target cells, but the IGF-system is not well-

described herein.

Objective

To perform a thorough comparison of the IGF-system in suction blister fluid (SBF) vs. in serum, with

emphasis on bioactive IGF levels.

Design

Eight hour study including samples collected in the fasting state (20 h) and after a meal.
http://www.sciencedirect.com/science/journal/10966374http://www.sciencedirect.com/science/journal/10966374http://www.sciencedirect.com/science/journal/10966374/22/6http://www.sciencedirect.com/science/journal/10966374/22/6http://www.sciencedirect.com/science/article/pii/S1096637412000676#item1http://www.sciencedirect.com/science/article/pii/S1096637412000676#item1http://www.sciencedirect.com/science/article/pii/S1096637412000676#item1http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676#af0020http://www.sciencedirect.com/science/article/pii/S1096637412000676#af0020http://www.sciencedirect.com/science/article/pii/S1096637412000676#af0020http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676#af0025http://www.sciencedirect.com/science/article/pii/S1096637412000676#af0025http://www.sciencedirect.com/science/article/pii/S1096637412000676#af0025http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676#af0020http://www.sciencedirect.com/science/article/pii/S1096637412000676#af0020http://www.sciencedirect.com/science/article/pii/S1096637412000676#af0020http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676#af0020http://www.sciencedirect.com/science/article/pii/S1096637412000676#af0020http://www.sciencedirect.com/science/article/pii/S1096637412000676#af0020http://dx.doi.org/10.1016/j.ghir.2012.08.002http://dx.doi.org/10.1016/j.ghir.2012.08.002http://www.sciencedirect.com/science/help/doi.htmhttp://www.sciencedirect.com/science/help/doi.htmhttp://www.sciencedirect.com/science/help/doi.htmhttp://www.sciencedirect.com/science?_ob=RedirectURL&_method=outwardLink&_partnerName=936&_eid=1-s2.0-S1096637412000676&_pii=S1096637412000676&_origin=article&_zone=art_page&_targetURL=https%3A%2F%2Fs100.copyright.com%2FAppDispatchServlet%3FpublisherName%3DELS%26contentID%3DS1096637412000676%26orderBeanReset%3Dtrue&_acct=C000228598&_version=1&_userid=12227422&md5=50991adca575eba99fc9408a2374209bhttp://www.sciencedirect.com/science?_ob=RedirectURL&_method=outwardLink&_partnerName=936&_eid=1-s2.0-S1096637412000676&_pii=S1096637412000676&_origin=article&_zone=art_page&_targetURL=https%3A%2F%2Fs100.copyright.com%2FAppDispatchServlet%3FpublisherName%3DELS%26contentID%3DS1096637412000676%26orderBeanReset%3Dtrue&_acct=C000228598&_version=1&_userid=12227422&md5=50991adca575eba99fc9408a2374209bhttp://www.sciencedirect.com/science?_ob=ShoppingCartURL&_method=add&_eid=1-s2.0-S1096637412000676&_acct=C000228598&_version=1&_userid=10&_ts=1360878423&md5=08f3aaffbdab27159b9846bcd9f70302http://www.sciencedirect.com/science?_ob=RedirectURL&_method=outwardLink&_partnerName=936&_eid=1-s2.0-S1096637412000676&_pii=S1096637412000676&_origin=article&_zone=art_page&_targetURL=https%3A%2F%2Fs100.copyright.com%2FAppDispatchServlet%3FpublisherName%3DELS%26contentID%3DS1096637412000676%26orderBeanReset%3Dtrue&_acct=C000228598&_version=1&_userid=12227422&md5=50991adca575eba99fc9408a2374209bhttp://www.sciencedirect.com/science/help/doi.htmhttp://dx.doi.org/10.1016/j.ghir.2012.08.002http://www.sciencedirect.com/science/article/pii/S1096637412000676#af0020http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676#af0020http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676#af0025http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676#af0020http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676http://www.sciencedirect.com/science/article/pii/S1096637412000676#item1http://www.sciencedirect.co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Setting

Clinical research facility.

Participants

Six healthy males (age 37.0 8.8 years, BMI 22.5 1.4 kg/m2) (mean SD).

Main outcome measure

Serum and SBF concentrations of bioactive IGF (determined in vitro by specific IGF-I receptor (IGF-

IR) phosphorylation assay), immunoreactive IGF and IGF binding protein (IGFBP) levels, Western

ligand blotting (WLB) of IGFBPs and IGFBP-3 Western immunoblotting (WiB).

Results

The ability of SBF to phosphorylate the IGF-IR in vitro was 41 27% higher than that of serum

(P= 0.007 by repeated measures ANOVA). By contrast, immunoreactive IGF and IGFBP-

concentrations were approximately 50% lower in SBF than in serum (all P 0.002). A marked

difference in the composition of IGFBPs between serum and SBF was observed, including 3-fold

elevated amounts of IGFBP-3 fragments in SBF (P< 0.001). For both IGF-I, IGF-II and IGFBP-2, the

effect of food intake differed between serum and SBF (all P 0.03).

Conclusion

Despite lower concentrations, the in vitro IGF bioactivity was higher in SBF than in serum. This may

relate to an increased enzymatic IGFBP-degradation and an altered IGFBP-composition in SBF,

making more IGF-I and -II accessible to the IGF-IR. The impact of food intake on the IGF system

differs between serum and interstitial fluid.

Keywords

Extracellular fluid; Insulin-like growth factor; Insulin-like growth factor binding protein; Biological assay; Kinase receptor activation assay; Fasting

Figures and tables from this article:


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Fig. 1. A. Vacuum pumps generate a suction of 300 mm Hg. B. Suction in progress with the reservoirs placed

on the abdominal skin at the level of the umbilicus. C. After the suction period, blisters are present on the skin.

The blister fluid is harvested with a syringe. A white box has been added in panel A to mask the underwear

brand name.

Figure options

Fig. 2. Concentrations or activities as a function of time for insulin, IGF-I, IGF-II, bioactive IGF, IGFBP-1,

IGFBP-2, IGFBP-3 and acid labile subunit (ALS). Dark circles and dashed lines represent mean suction blister

fluid values, whereas dark squares and solid lines represent mean serum values. Light gray lines represent

individual observations. Statistically significant differences by ANOVA are given as follows: *C*: different level

of lines, i.e. Pcompartment < 0.05, *M*: slopes of lines differ from zero, i.e. Pmeal < 0.05, and *I*: different slopes of

lines in the two compartments, i.e. P interaction < 0.05. The respective P-values are given inTable 1.

Figure options

Fig. 3. Western immune blotting (WiB) and western ligand blotting (WLB) data given as mean + SD. Lightly

colored bars represent serum and dark bars represent suction blister fluid. S = serum, B = suction blister fluid.

1 = fasting, 2 = following the meal. A: Fraction of proteolyzed IGFBP-3 by WiB. In SBF, significantly more

IGFBP-3 was proteolyzed as compared to serum (P< 0.001). The two lanes from patient number 4 are from

the same gel. B: Relative distribution of labeled IGF-I by WLB grouped by WLB band for each band, there

are 4 columns, representing serums 1 and 2 and blisters 1 and 2. The bands have previously been identified

as IGFBP-3 (approximately 3842 kDa), IGFBP-2 (approximately 32 kDa), IGFBP-1, glycosylated IGFBP-4,

IGFBP-5 and IGFBP-6 (approximately 28 kDa) and finally nonglycosylated IGFBP-4 (approximately 24 kDa).

In SBF, significantly less labeled IGF-I was bound to the IGFBP-3 bands when compared to serum (P= 0.004).

The insert shows a representative WLB gel.

Figure options
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Table 1. Results and statistics.

Measurements and the corresponding statistical inference. 1: ANOVA intra-subject term in a repeated

measures model. 2: ANOVA interaction term between meal and compartment. IfP> 0.10, interaction term

omitted from the model, denoted by . WLB: Western Ligand Blot. WLB 14 represent different bands on the

WLB gel, previously identified as: IGFBP-1, glycosylated IGFBP-4, IGFBP-5 and IGFBP-6 (WLB 1,

approximately 28 kDa), IGFBP-2 (WLB 2, approximately 32 kDa), and IGFBP-3 (WLB 3, approximately 38

42 kDa) and nonglycosylated IGFBP-4 (WLB 4, approximately 24 kDa). Values are relative to total amount of

labeled IGF-1 bound for each sample. WiB: Western immunoblotting of IGFBP-3. For WiB, only fasting values

were available, excluding mealand interaction terms from the model. Insulin in pmol/L. IGF-I, IGF-II, KIRA,

IGFBP-1, IGFBP-2, IGFBP-3 in g/L. ALS in units/L. WLB 1-4 and WiB in % of total. P-values of less than 5%

are printed in bold.


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Hormon Pertumbuhan & Penelitian IGF

Volume 22, Issue 6, Desember 2012, Pages 234-239

Cairan interstitial mengandung tinggi dalam bioaktivitas IGF vitro

dibandingkan serum: Sebuah studi menggunakan teknik melepuh

hisap

Ulrick Espelunda, b,,,

Klaus Sndergaardc, d,

Peter Bjerringe,

Allan Flyvbjerga, b,

Jan Frystyka, b

Departemen Endokrinologi dan Internal Medicine, Aarhus University Hospital, Denmark

b Laboratorium Penelitian Medis, Departemen Kedokteran Klinis, Universitas Aarhus, Aarhus C, Denmark

c Departemen Rheumatology, Aarhus University Hospital, Denmark

d Departemen Kedokteran Klinis, Universitas Aarhus, Aarhus C, Denmark

e Mlholm Penelitian, Mlholm Rumah Sakit Swasta A / S, Vejle, Denmark

Abstrak

Konteks

Beredar insulin-seperti faktor pertumbuhan (IGFs) terikat dalam kompleks yang mempengaruhi

mereka jaringan-aksesibilitas. Cairan interstitial yang dekat dengan sel target, tetapi IGF-sistem tidak

baik dijelaskan di sini.

Tujuan

Untuk melakukan perbandingan menyeluruh dari sistem IGF-dalam cairan suction blister (SBF) vs

dalam serum, dengan penekanan pada tingkat IGF bioaktif.

Disain

Delapan jam studi termasuk sampel yang dikumpulkan dalam keadaan puasa (20 jam) dan setelahmakan.


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Mengatur

Klinis fasilitas penelitian.

Peserta

Enam laki-laki yang sehat (usia 37,0 8,8 tahun, BMI 22,5 1,4 kg/m2) (mean SD).

Main hasil mengukur

Serum dan SBF konsentrasi IGF bioaktif (in vitro ditentukan oleh spesifik reseptor IGF-I (IGF-IR) assay

fosforilasi), immunoreactive IGF dan mengikat protein IGF (IGFBP) tingkat, ligan Western blotting

(WLB) dari IGFBPs dan IGFBP-3 Barat imunoblotting (WIB).

Hasil

Kemampuan SBF untuk memfosforilasi IGF-IR in vitro adalah 41 27% lebih tinggi dari serum (P =

0,007 dengan tindakan berulang ANOVA). Sebaliknya, IGF immunoreactive dan IGFBP-konsentrasi

adalah sekitar 50% lebih rendah daripada di dalam SBF serum (semua P 0,002). Perbedaan ditandai

dalam komposisi IGFBPs antara serum dan SBF diamati, termasuk 3 kali lipat jumlah peningkatan

IGFBP-3 fragmen dalam SBF (P


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Gambar dan tabel dari artikel ini:

Gambar. 1. Pompa vakum A. menghasilkan hisap dari 300 mm Hg. B. Suction berlangsung dengan

reservoir ditempatkan pada kulit perut pada tingkat umbilikus. C. Setelah masa hisap, lepuh yang

hadir pada kulit. Cairan blister dipanen dengan jarum suntik. Sebuah kotak putih telah ditambahkan

di panel A untuk menutupi nama merek pakaian.

Gambar pilihan

Gambar. 2. Konsentrasi atau kegiatan sebagai fungsi waktu untuk insulin, IGF-I, IGF-II, bioaktif IGF,

IGFBP-1, IGFBP-2, IGFBP-3 dan asam subunit labil (ALS). Lingkaran hitam dan garis putus-putus

mewakili nilai-nilai melepuh hisap cairan berarti, sedangkan kotak gelap dan garis padat mewakili

nilai-nilai serum. Garis abu-abu terang mewakili pengamatan individu. Perbedaan signifikan secara

statistik dengan ANOVA diberikan sebagai berikut: * C *: tingkat yang berbeda dari garis, yaitu

Pcompartment


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melepuh. S = serum, B = hisap cairan melepuh. 1 = puasa, 2 = setelah makan. A: Fraksi dari

proteolyzed IGFBP-3 oleh Wib. Dalam SBF, secara signifikan lebih IGFBP-3 ini proteolyzed

dibandingkan dengan serum (P 0,10,interaksi jangka dihilangkan dari model, dinotasikan dengan "-". WLB: Western Blot Ligan. WLB 1-4

merupakan band yang berbeda pada gel WLB, yang sebelumnya diidentifikasi sebagai: IGFBP-1,

glikosilasi IGFBP-4, IGFBP-5 dan IGFBP-6 (WLB 1, sekitar 28 kDa), IGFBP-2 (WLB 2, sekitar 32 kDa),

dan IGFBP-3 (WLB 3, sekitar 38-42 kDa) dan nonglycosylated IGFBP-4 (WLB 4, sekitar 24 kDa). Nilai

adalah relatif terhadap jumlah total berlabel IGF-1 terikat untuk setiap sampel. Wib: Western

imunoblotting dari IGFBP-3. Untuk Wib, hanya nilai-nilai puasa yang tersedia, termasuk istilah makan

dan interaksi dari model. Insulin di pmol / L. IGF-I, IGF-II, KIRA, IGFBP-1, IGFBP-2, IGFBP-3 di ug / L.

ALS di unit / L. WLB 1-4 dan WIB di% dari total. P-nilai kurang dari 5% yang dicetak dengan huruf

tebal.

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