Establishment and Primary Application of a Highly‐Sensitive Orexin‐A Radioimmunoassay

Abstract

Orexin-A was labeled by 125I using the chloramine-T method, and was purified with a Sephadex G-25 chromatographic column. The reaction between antigen and antibody was carried out by a one-step balance method and was incubated at 4 degrees C for 24 hours, then bonded and free antigen were separated by PR reagent. The detection range of this RIA is 21-2000 pg/mL; the lowest detection level is 21 pg/mL. The intra-assay and inter-assay variations were 7.8% and 9.7%, respectively. Plasma orexin-A levels of 30 normal individuals and 30 patients with hyperlipidemia (serum triglyceride > 1.7 mmol/L and serum total cholesterol > 5.7 mmol/L) were detected by this RIA, while orexin-A levels of plasma and hypothalamus in rat intestinal ischemia-reperfusion injury model were also measured. Plasma orexin-A levels of normal individuals was 338.48 +/- 20.24 pg/mL, while those of patients with hyperlipidemia were 343.51 +/- 15.49 pg/mL; there were no significant differences between these two groups t = -0.1976; P = 0.8441. We also found that orexin-A levels of rat plasma and hypothalamus did not express a significant change during the early stages of intestinal ischemia-reperfusion injury. These results have shown that this orexin-A radioimmunoassay is stable, simple, and specific, being sensitive enough to test orexin-A levels in human plasma, rat plasma, and hypothalamus.

Both orexin receptors are expressed in rat ovaries and fluctuate with the estrous cycle: effects of orexin receptor antagonists on gonadotropins and ovulation

“…Several hypotheses can be formulated to explain the presence of both receptors in the ovary in the absence of PPO; for example, the orexinergic peptide may originate elsewhere or the receptors may be activated by other molecules present in the gonad. The possibility that orexins may originate from outside the ovary and arrive by circulation is in line with the fact that immunoreactive orexin A has been described in human and rat plasma (2,36). In this regard, PPO has been shown to be localized in discrete neurons in the lateral, perifornical, and dorsomedial hypothalamus (45).…”

Effect of intestinal ischemia/reperfusion injury on leptin and orexin-A levels

“…Standard leptin and serum samples were mixed with 100 µL leptin antibody and 100 µL 125 I-leptin (about 20,000 cpm), while standard orexin-A and plasma samples were mixed with 50 µL orexin-A antibody and 50 µL 125 I-orexin-A (about 20,000 cpm), and they were allowed to react at 4°C for 24 h. After addition of 500 µL immune separating reagent and reaction for 15 min at room temperature, all tubes were centrifuged at 3,000xg for 20 min at 4°C. Then the supernatant was discarded and radioactivity of precipitates in the tubes was detected to plot a standard curve [9,10]. Before testing, Coomassie brilliant blue G-250 method [13] was used to determine the total protein concentration in the tissue homogenate, and adipose leptin levels (ng) or hypothalamus orexin-A levels (pg) were compared in terms of 100 µg total protein of each tissue sample.…”

“…We have developed highly sensitive radioimmunoassays for rat leptin and orexin-A [9,10], which were used to detect the changes of leptin and orexin-A levels in peripheral blood (serum and plasma) and central secretory tissues (adipose tissue and hypothalamus) during intestinal I/R injury in rats. Moreover, we employed reverse transcriptase-PCR to detect mRNA expressions of leptin and orexin-A in adipose tissue and hypothalamus after the injury respectively, with an attempt to understand the roles of leptin and orexin-A in the acute inflammatory responses.…”