We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases (e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G(1) cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT.

IGF-I induz proliferação e diferenciação celular. Neste estudo visamos sua participação na imunidade específica na leishmaniose. Estudamos efeito de citocinas Th1 (IFN-g) e Th2 (IL- 4 mais IL-13) na modulação da produção de IGF-I e seu reflexo no parasitismo de macrófago (MØ) de camundongos BALB/c e C57BL/6, infectados por amastigotas e promastigotas de Leishmania (L.) major. Iniciamos avaliando os efeitos de IGF-I no modelo, onde no desenvolvimento da lesão IGF-I induziu aumento maior de lesão em camundongos BALB/c. Nos camundongos C57BL/6 o efeito foi marcante com evolução progressiva da lesão enquanto que sem o fator, a lesão se controlava. Como a produção de IGF-I por MØ é modulada por citocinas, onde IFN-g diminui e IL-4 e IL-13 aumentam a sua expressão, fomos estudar seus efeitos no modelo proposto onde MØ foram infectados com amastigotas ou promastigotas de L.(L.) major (parasitos/célula = 2:1) e incubados com IFN-g (200U/mL) ou IL-4 (2ng/mL) mais IL-13 (5ng/mL). IFN-g induziu aumento significante na produção de NO nos dois modelos estudados. O efeito de citocinas Th1 e Th2 sobre o parasitismo em MØ foi distinto em BALB/c e C57BL/6. Quando infectados por amastigotas, o parasitismo aumentou sob estímulo com IL-4 mais IL-13 e diminuiu com IFN-g como era esperado. No entanto, quando infectado por promastigota, o parasitismo aumentou com IL-4 mais IL-13 somente em células BALB/c. Por outro lado, o parasitismo diminuiu com IFN- g somente em células C57BL/6. Mesmo na ausência do estímulo por citocinas, observou-se aumento na expressão de RNA de IGF-I nos MØ de animais BALB/c infectados por formas amastigotas ou promastigotas e de C57BL/6 infectados por promastigotas. Um resultado destoante foi à diminuição da expressão de IGF-I em MØ de C57BL/6 infectados por amastigotas. Em células BALB/c, essa expressão aumentada de IGF-I ocorrida em função da infecção, somente se alterou com IL-4 mais IL-13 quando infectadas por amastigotas. Em células C57BL/6, a expressão aumentada de IGF-I com a infecção por prormastigota, sofreu diminuição sob estímulo com IFN-g, não se alterando com citocinas Th2. A expressão diminuída de IGF-I com a infecção por amastigotas diminuiu ainda mais com IFN-g e aumentou com citocinas TH2. Os dados sugerem a possibilidade de modulação do efeito de citocinas pela expressão diferenciada de IGF-I em macrófagos infectados por L. (L.) major / Insulin-like growth factor (IGF)-I induces cell proliferation and differentiation. In this study, we focused on its participation on specific immunity. We studied the effect of Th1 (IFN-g) and Th2 (IL-4 plus IL-13) cytokines on the modulation of the production of IGF-I and its influence on BALB/c and C57BL/6 macrophage (MØ) parasitism using Leishmania (L.) major amastigote and promastigote. IGF-I expression was increased in infected macrophages. Initially, we substantiated the effect of IGF-I on in vitro growth of Leishmania (L.) major promastigote, on cutaneous lesion development in vivo and diminished production of nitric oxide in MØ. On development of the lesion, IGF-I induced greater increase of the lesion in BALB/c mice. In C5BL/6 mice, its effect was more pronounced with progression of the lesion whilst without the factor it was controlled. Since IGF-I production by MØ is modulated by cytokines where IFN-g decreases and IL-4 plus IL-13 increase its expression, we studied their effects in BALB/c and C57BL/6 MØ. Macrophages were infected with L.(L.) major amastigotes or promastigotes (parasites/cell = 2:1) and incubated with IFN-g (200U/mL) or IL-4 (2ng/mL) plus IL-13 (5ng/mL). IFN-g induced a significant increase of NO production by MØ from both models. The effect of Th1 and Th2 cytokines on parasitism was distinct when MØ infected with amastigotes, the parasitism increased upon IL-4 plus IL-13 stimuli and diminished with IFN-g as expected. However, when infected with promastigotes, the parasitism increased with IL-4 plus IL-13 only in BALB/c cells. Conversely, the parasitism diminished with IFN-g only in C57BL/6. Even in the absence of cytokine stimuli, an increase of the expression of IGF-I mRNA was observed in L (L.) major amastigote- and promastigote- infected BALB/c MØ and in promastigoteinfected C57BL/6 MØ. A dissonant result was a diminished IGF-I expression in amastigoteinfected C57BL/6 MØ. In BALB/c this increased IGF-I expression with infection only altered with IL-4 plus IL-13 in amastigote-infected cells. In C57BL/6 cells, the increased IGF-I expression upon prormastigote infection diminished under IFN-g stimulus that was not altered with Th2 cytokines. A decreased IGF-I expression with amastigote-infection diminished even more with IFN-g and increased with Th2 cytokines. These data suggest a possibility of modulation of the effect of cytokines on distinct expression of IGF-I in L. (L.) major-infected MØ

Common measures of egg quality have been survival to specific developmental stages, higher hatching rate of fertilized eggs and final production of fry. Determinants of egg quality are variable among and between teleost species and no common unified criteria have been established. Maternally inherited genes influence egg quality and early embryo development is partially programmed by the messenger ribonucleic acid (mRNA). Among the genes, the insulin family is important for growth functions and the presence of their transcripts in the ovary, oocytes and embryos implies their involvement during the reproductive process and their relevance to egg quality. The insulin-like growth factor (IGF) system has three components, the ligands IGF-I and II, the IGFBPs (insulin-like growth factor binding proteins) and the IGF receptors that mediate biological activity of the ligands. Vitellogenin (Vtg) is the major source of nutrients for the developing embryo and elevated levels in female fish plasma signals gonadal development preceding spawning. In oviparous fish where the developing embryo is dependent on the stored food in the yolk, vitellogenin levels in the egg could indicate its capability to support embryonic growth. This study aimed to develop molecular tools, specifically probes for IGF-I, IGF-II and IGF-IR, for the evaluation of fish egg quality. These probes would be used to determine expression levels of IGF-I, IGF-II and IGF-IR during egg development to assess their potential as molecular indicators for egg quality. In addition, this study also aimed to establish an enzyme-linked immunoassay (ELISA) for quantifying Vtg in fish eggs and determine if differences in Vtg levels could be linked to fertilization and hatching success. Through reverse-transcription polymerase chain reaction (RT-PCR) putative complementary deoxyribonucleic acid (cDNA) fragments of IGF-I, IGF-II and IGF-IR were cloned and sequenced from mullet (Mugil cephalus) and grouper (Epinephelus coioides). The relative expression ratio of the three genes in the eggs of mullet and grouper were assayed by quantitative PCR (QPCR) and calculated using the Pfaffl method (Pfaffl, 2001). Levels of vitellogenin in different batches of mullet eggs were quantified by ELISA. Spawned eggs of grouper were grouped into low (<60%) or high (>60%) fertilization rate (FR) and the fertilized eggs that were incubated until hatching were grouped into medium (>90%) or high (>90%) hatching rate (HR). Samples were categorized into sinking eggs, late embryo and hatched larvae. Relative expression ratio of IGF-II was significantly high (P<0.01) compared to IGF-I and IGF-IR in all samples examined. All three genes were strongly expressed in sinking eggs compared to either late embryo or hatched larvae. However, there was no significant interaction effect between the genes and the samples analyzed. Mullet samples all came from a high FR and high HR group and were categorized into sinking, multicell stage, blastula, gastrula, late embryo and hatched larvae. There was a significant interaction effect (P<0.01) between gene and stage, showing that genes are differentially expressed during embryonic development. IGF-II was strongly expressed relative to the other genes in all stages examined and was highest during the gastrula stage. Vtg levels were examined in mullet oocytes and egg samples that were grouped into 4; oocytes from females that subsequently spawned, had fertilized eggs which hatched (Group A); oocytes from females that did not spawn, therefore no fertilization and no hatching (Group B); eggs that were stripped, artificially fertilized but no hatching (Group C); and eggs that were spawned, assumed to be fertilized but did not hatch (Group D). Group A showed a trend of higher Vtg levels than the other three but this result was not statistically significant.

IGF-I induz proliferação e diferenciação celular. Neste estudo visamos sua participação na imunidade específica na leishmaniose. Estudamos efeito de citocinas Th1 (IFN-g) e Th2 (IL- 4 mais IL-13) na modulação da produção de IGF-I e seu reflexo no parasitismo de macrófago (MØ) de camundongos BALB/c e C57BL/6, infectados por amastigotas e promastigotas de Leishmania (L.) major. Iniciamos avaliando os efeitos de IGF-I no modelo, onde no desenvolvimento da lesão IGF-I induziu aumento maior de lesão em camundongos BALB/c. Nos camundongos C57BL/6 o efeito foi marcante com evolução progressiva da lesão enquanto que sem o fator, a lesão se controlava. Como a produção de IGF-I por MØ é modulada por citocinas, onde IFN-g diminui e IL-4 e IL-13 aumentam a sua expressão, fomos estudar seus efeitos no modelo proposto onde MØ foram infectados com amastigotas ou promastigotas de L.(L.) major (parasitos/célula = 2:1) e incubados com IFN-g (200U/mL) ou IL-4 (2ng/mL) mais IL-13 (5ng/mL). IFN-g induziu aumento significante na produção de NO nos dois modelos estudados. O efeito de citocinas Th1 e Th2 sobre o parasitismo em MØ foi distinto em BALB/c e C57BL/6. Quando infectados por amastigotas, o parasitismo aumentou sob estímulo com IL-4 mais IL-13 e diminuiu com IFN-g como era esperado. No entanto, quando infectado por promastigota, o parasitismo aumentou com IL-4 mais IL-13 somente em células BALB/c. Por outro lado, o parasitismo diminuiu com IFN- g somente em células C57BL/6. Mesmo na ausência do estímulo por citocinas, observou-se aumento na expressão de RNA de IGF-I nos MØ de animais BALB/c infectados por formas amastigotas ou promastigotas e de C57BL/6 infectados por promastigotas. Um resultado destoante foi à diminuição da expressão de IGF-I em MØ de C57BL/6 infectados por amastigotas. Em células BALB/c, essa expressão aumentada de IGF-I ocorrida em função da infecção, somente se alterou com IL-4 mais IL-13 quando infectadas por amastigotas. Em células C57BL/6, a expressão aumentada de IGF-I com a infecção por prormastigota, sofreu diminuição sob estímulo com IFN-g, não se alterando com citocinas Th2. A expressão diminuída de IGF-I com a infecção por amastigotas diminuiu ainda mais com IFN-g e aumentou com citocinas TH2. Os dados sugerem a possibilidade de modulação do efeito de citocinas pela expressão diferenciada de IGF-I em macrófagos infectados por L. (L.) major / Insulin-like growth factor (IGF)-I induces cell proliferation and differentiation. In this study, we focused on its participation on specific immunity. We studied the effect of Th1 (IFN-g) and Th2 (IL-4 plus IL-13) cytokines on the modulation of the production of IGF-I and its influence on BALB/c and C57BL/6 macrophage (MØ) parasitism using Leishmania (L.) major amastigote and promastigote. IGF-I expression was increased in infected macrophages. Initially, we substantiated the effect of IGF-I on in vitro growth of Leishmania (L.) major promastigote, on cutaneous lesion development in vivo and diminished production of nitric oxide in MØ. On development of the lesion, IGF-I induced greater increase of the lesion in BALB/c mice. In C5BL/6 mice, its effect was more pronounced with progression of the lesion whilst without the factor it was controlled. Since IGF-I production by MØ is modulated by cytokines where IFN-g decreases and IL-4 plus IL-13 increase its expression, we studied their effects in BALB/c and C57BL/6 MØ. Macrophages were infected with L.(L.) major amastigotes or promastigotes (parasites/cell = 2:1) and incubated with IFN-g (200U/mL) or IL-4 (2ng/mL) plus IL-13 (5ng/mL). IFN-g induced a significant increase of NO production by MØ from both models. The effect of Th1 and Th2 cytokines on parasitism was distinct when MØ infected with amastigotes, the parasitism increased upon IL-4 plus IL-13 stimuli and diminished with IFN-g as expected. However, when infected with promastigotes, the parasitism increased with IL-4 plus IL-13 only in BALB/c cells. Conversely, the parasitism diminished with IFN-g only in C57BL/6. Even in the absence of cytokine stimuli, an increase of the expression of IGF-I mRNA was observed in L (L.) major amastigote- and promastigote- infected BALB/c MØ and in promastigoteinfected C57BL/6 MØ. A dissonant result was a diminished IGF-I expression in amastigoteinfected C57BL/6 MØ. In BALB/c this increased IGF-I expression with infection only altered with IL-4 plus IL-13 in amastigote-infected cells. In C57BL/6 cells, the increased IGF-I expression upon prormastigote infection diminished under IFN-g stimulus that was not altered with Th2 cytokines. A decreased IGF-I expression with amastigote-infection diminished even more with IFN-g and increased with Th2 cytokines. These data suggest a possibility of modulation of the effect of cytokines on distinct expression of IGF-I in L. (L.) major-infected MØ

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19399号 / 医博第4050号 / 新制||医||1012(附属図書館) / 32424 / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 川口 義弥, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Sabe-se que o hipotireoidismo (Hipo) resulta em supressão do eixo hormônio de crescimento (GH)/ insulin-like growth factor I (IGF-I) e em atraso no desenvolvimento esquelético. Em um estudo anterior, vimos que o tratamento de ratas jovens Hipo com GC-1, um análogo da triiodotironina (T3) seletivo pela isoforma <font face=\"symbol\">b de receptor de hormônio tireoideano (TR<font face=\"symbol\">b), não teve efeito sobre o IGF-I sérico ou sobre a expressão protéica de IGF-I nas lâminas epifisiais, mas parcialmente reverteu alterações esqueléticas decorrentes do Hipo, o que sugere que: (i) o desenvolvimento esquelético requer ações do T3 mediadas pelo TR<font face=\"symbol\">a1 e TR<font face=\"symbol\">b1 (isoformas de TR expressas no osso); ou (ii) requer interações entre o eixo GH/IGF-I e o hormônio tireoideano. Neste estudo, investigamos essas hipóteses tratando ratas recém desmamadas Hipo com T3 ou GC-1 em associação ou não com o GH por 4 semanas. Os nossos achados mostram que o T3 e GH interagem para promover o desenvolvimento ósseo, mas que uma série de efeitos do T3 nesse processo independe do eixo GH/IGF-I e são mediadas pelo TR<font face=\"symbol\">a e/ou TR<font face=\"symbol\">b. / Thyroid hormone (TH) has important effects on bone development and metabolism. It is known that triiodotyronine (T3) has indirect actions in the skeleton through its influence on the production and secretion of growth hormone (GH)/ insulin-like growth factor (IGF-I) and/or other factors. On the other hand, direct actions of T3 on bone are recognized but not yet clear. Most of T3 action is mediaded by its nuclear receptors (TRs). TR<font face=\"symbol\">a1, TR<font face=\"symbol\">b1 e TR<font face=\"symbol\">b2 bind T3, while TR<font face=\"symbol\">a2 does not bind T3 and acts as an antagonist of genic transcription of TR<font face=\"symbol\">a1 and TR<font face=\"symbol\">b1. All these receptors, except TR<font face=\"symbol\">b2, are expressed in chondrocytes of growth plate, osteoblasts and osteoclastos. However, the functional roles of each TR isoformas in the bone development are incompletely understood. A few years, it is development GC-1, a synthetic analog of T3 which is selectivwe for TR<font face=\"symbol\">b1 over TR<font face=\"symbol\">a1. In recent study, we showed that treatment of hypothyroid young rats with T3 revert the IGF-I deficiency and skeleton defects caused by hypothyroidism. Since GC-1 treatment does not effects on serum levels of IGF-I or protein expression of IGF-I in the growth plate, but revert some bone alterations induced by T3 deficiency. Considering the selectivity of GC-1 for TR<font face=\"symbol\">b, these findings suggest that T3 has effects on bone development that are mediated by TR<font face=\"symbol\">b and independent of GH/IGF-I axis. On the other hand, the inability of GC-1 in completely revert the alterations of bone development suggests that the normal skeleton development require (i) T3 actions mediated by TR<font face=\"symbol\">a1 and TR<font face=\"symbol\">b1, or (ii) synergic or additive actions between GH/IGF-I axis and thyroid hormone. To investigate these hypotheses, 21 day-old hypothyroid female rats were treated with T3 or GC-1 in association or not with GH for 4 weeks. Our findings show that T3 interacts with GH to promote body growth, differentiation of growth plate hypertrofic chondrocytes, intramembranous ossification of cranial bone, and increased of bone resistance and other biomechanics parameters that contribute to the best bone quality. On the other hand, ours results suggest strongly that TH acts in bone mass acquisition, in organization of growth plate chondrocytes and endocondral ossification mainly independent of GH/IGF-I axis and via TR<font face=\"symbol\">a and/or TR<font face=\"symbol\">b.

Sabe-se que o hipotireoidismo (Hipo) resulta em supressão do eixo hormônio de crescimento (GH)/ insulin-like growth factor I (IGF-I) e em atraso no desenvolvimento esquelético. Em um estudo anterior, vimos que o tratamento de ratas jovens Hipo com GC-1, um análogo da triiodotironina (T3) seletivo pela isoforma <font face=\"symbol\">b de receptor de hormônio tireoideano (TR<font face=\"symbol\">b), não teve efeito sobre o IGF-I sérico ou sobre a expressão protéica de IGF-I nas lâminas epifisiais, mas parcialmente reverteu alterações esqueléticas decorrentes do Hipo, o que sugere que: (i) o desenvolvimento esquelético requer ações do T3 mediadas pelo TR<font face=\"symbol\">a1 e TR<font face=\"symbol\">b1 (isoformas de TR expressas no osso); ou (ii) requer interações entre o eixo GH/IGF-I e o hormônio tireoideano. Neste estudo, investigamos essas hipóteses tratando ratas recém desmamadas Hipo com T3 ou GC-1 em associação ou não com o GH por 4 semanas. Os nossos achados mostram que o T3 e GH interagem para promover o desenvolvimento ósseo, mas que uma série de efeitos do T3 nesse processo independe do eixo GH/IGF-I e são mediadas pelo TR<font face=\"symbol\">a e/ou TR<font face=\"symbol\">b. / Thyroid hormone (TH) has important effects on bone development and metabolism. It is known that triiodotyronine (T3) has indirect actions in the skeleton through its influence on the production and secretion of growth hormone (GH)/ insulin-like growth factor (IGF-I) and/or other factors. On the other hand, direct actions of T3 on bone are recognized but not yet clear. Most of T3 action is mediaded by its nuclear receptors (TRs). TR<font face=\"symbol\">a1, TR<font face=\"symbol\">b1 e TR<font face=\"symbol\">b2 bind T3, while TR<font face=\"symbol\">a2 does not bind T3 and acts as an antagonist of genic transcription of TR<font face=\"symbol\">a1 and TR<font face=\"symbol\">b1. All these receptors, except TR<font face=\"symbol\">b2, are expressed in chondrocytes of growth plate, osteoblasts and osteoclastos. However, the functional roles of each TR isoformas in the bone development are incompletely understood. A few years, it is development GC-1, a synthetic analog of T3 which is selectivwe for TR<font face=\"symbol\">b1 over TR<font face=\"symbol\">a1. In recent study, we showed that treatment of hypothyroid young rats with T3 revert the IGF-I deficiency and skeleton defects caused by hypothyroidism. Since GC-1 treatment does not effects on serum levels of IGF-I or protein expression of IGF-I in the growth plate, but revert some bone alterations induced by T3 deficiency. Considering the selectivity of GC-1 for TR<font face=\"symbol\">b, these findings suggest that T3 has effects on bone development that are mediated by TR<font face=\"symbol\">b and independent of GH/IGF-I axis. On the other hand, the inability of GC-1 in completely revert the alterations of bone development suggests that the normal skeleton development require (i) T3 actions mediated by TR<font face=\"symbol\">a1 and TR<font face=\"symbol\">b1, or (ii) synergic or additive actions between GH/IGF-I axis and thyroid hormone. To investigate these hypotheses, 21 day-old hypothyroid female rats were treated with T3 or GC-1 in association or not with GH for 4 weeks. Our findings show that T3 interacts with GH to promote body growth, differentiation of growth plate hypertrofic chondrocytes, intramembranous ossification of cranial bone, and increased of bone resistance and other biomechanics parameters that contribute to the best bone quality. On the other hand, ours results suggest strongly that TH acts in bone mass acquisition, in organization of growth plate chondrocytes and endocondral ossification mainly independent of GH/IGF-I axis and via TR<font face=\"symbol\">a and/or TR<font face=\"symbol\">b.

Tillväxthormon och insulinliknande tillväxtfaktor-1 är blodprover som tas vid misstanke om akromegali. Immulite 2000 XPi är ett instrument som använder chemiluminescent microparticle immunoassay metodik för att kvantitativt mäta koncentrationen av GH och IGF-1. Analys av biokemiska markörer påverkas av flera olika faktorer. En viktig sådan faktor är hemolys. Hemolys innebär att de röda blodkropparna går sönder, vilket leder till frisättning av dess innehåll såsom hemoglobin i serum/plasma. Syftet med detta projekt var att undersöka hur hemolys påverkar resultatet av GH- och IGF-1-prover på immulite 2000 XPi. Utifrån erhållna resultat kommer klinisk kemi vid Universitetssjukhuset Örebros laboratorie rutiner vid hemolytiska prover på GH eller IGF-1 att uppdateras . Hemolys tillverkades och tillsattes till olika serumprover med låg och hög nivå av GH respektive IGF-1 för att erhålla prover med varierande grad av hemolys. Resultatet visade att ökande hemolysindex korrelerar med GH- respektive IGF-1-koncentrationer, med undantag för den låga GH koncentrationen som inte uppvisade någon korrelation till hemolysindex. En 10%:ig skillnad av GH och IGF-1- koncentrationer uppnåddes vid en ökning av hemolysindex med 555,40 mg/dL för GH respektive 333,3 mg/dL för IGF-1.Utifrån resultaten var hemolysgränsen likvärdig med tillverkarens gränser. Därför kan klinisk kemi på Universitetssjukhuset Örebro fortsätta med nuvarande hemolysgränser. / Growth hormone and insulin-like growth factor-1 are blood samples taken upon suspicion of acromegaly and for follow-up of acromegaly treatment. Immulite 2000 XPi is an instrument that uses chemiluminescent microparticle immunoassay method to quantitatively measure the concentration of GH and IGF-1. Analysis of biochemical markers is affected by several different factors. An important such factor is hemolysis. Hemolysis means that the red blood cells break, whose contents leak into the serum/plasma. The study aimed to investigate how hemolysis affects the results of GH and IGF-1 samples on the Immulite 2000 XPi. The results will determine the routines for hemolytic samples for GH or IGF-1 at Örebro University Hospital. Hemolysis was produced and added to various serum samples with low and high levels of GH and IGF-1 to obtain samples with varying degrees of hemolysis. The results showed that increasing hemolysis index correlates with GH and IGF-1 concentrations, with the exception of low GH concentration, which did not show any correlation to hemolysis index. A 10% difference in GH and IGF-1 concentrations was achieved with an increase in hemolysis index of 555.40 mg/dL for GH and 333.3 mg/dL for IGF-1. Based on the results, the hemolysis limit was equivalent to the manufacturer's limits. Therefore, clinical chemistry at Örebro University Hospital can continue with current hemolysis limits.