Hypocretin Measures In Blood

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There is considerable controversy as to the usefulness of hypocretin measures in the blood. As stated earlier, we could not detect any hypocretin signals in human blood. (From 20 mL of plasma, the estimated level was less than 2.5 pg/mL). Similarly, reported findings based on blood measures are very controversial. In a typical example, a significant positive correlation between plasma hypocretin levels and apnea hypoxia index (AHI) was reported in obstructive sleep apnea patients (16). Another group reported a completely opposite result (i.e., significant negative correlations between AHI and blood hypocretin levels) (15) (see also ref. 24), whereas two other studies demonstrated no changes in CSF hypocretin levels in obstructive sleep apnea patients (7,25).

Regarding blood hypocretin status in narcolepsy, one group reported that there were no changes in plasma hypocretin levels in narcolepsy-cataplexy subjects (with CSF hypocretin deficiency) (9). Others reported significantly low blood hypocretin levels in narcolepsy (11/12 cases with cataplexy), although the sensitivity of low plasma hypocretin levels for narcolepsy was only 25% at 100% specificity (14).

At the conceptual level, hypocretins are likely to exist in the blood. Small amounts of pep-tides can leak from the brain to the periphery through the blood-brain barrier. An increasing number of studies have demonstrated that hypocretin receptors are present in the periphery (26) and that low levels of hypocretins are likely to be produced peripherally (27). All these factors can contribute to the existence of a small amount of hypocretin in plasma. If most of the hypocretin signal in the blood originates from the brain, and/or global (i.e., both central and peripheral) hypocretin deficiency exists in narcolepsy-cataplexy, then blood hypocretin measures may provide additional, but less invasive, diagnostic information.

We also observed reduced hypocretin signals in the blood in genetically produced hypocretin/orexin-deficient mice (i.e., orexin/ataxin-3 transgenic mice [TG] and prepro-orexin knockout [KO] (28). Several investigators, including us, had noted that rodents have relatively high hypocretin signals in the blood. This may be partially related to higher hypocretin levels in the CSF in these species (CSF hypocretin levels in rats are about five times higher than those in humans or dogs) (29).

Orexin Deficiency

Fig. 2. Serum hypocretin levels in prepro-orexin/ataxin-3 transgenic (TG) and wild-type (WT) mice and prepro-orexin knockout (KO) mice. (A,B) Hypocretin-1 levels were similar in males and females. Blood hypocretin levels were significantly lower in TG narcoleptic mice compared with the WT mice group (17.6 ± 1.6 pg/mL vs 35.8 ± 1.8 pg/mL, p < 0.0001). Thirty of 100 TG mice had undetectably low blood hypocretin levels, whereas only 1 of 96 WT mice had undetectable levels. The specificity of undetectable blood levels in TG mice was high (99%), although the sensitivity was only 30%. (C) Similarly, blood hypocretin levels were significantly lower in KO narcoleptic mice compared with a WT mice group (26.1 ± 3.0 pg/mL vs 36.3 ± 2.6 [SEM] pg/mL, p = 0.015). However, the overlap in hypocretin values between KO and WT mice was larger than that of the TG mice comparison. Four of 23 KO mice and 3 of 21 WT mice showed undetectable blood hypocretin levels.

Fig. 2. Serum hypocretin levels in prepro-orexin/ataxin-3 transgenic (TG) and wild-type (WT) mice and prepro-orexin knockout (KO) mice. (A,B) Hypocretin-1 levels were similar in males and females. Blood hypocretin levels were significantly lower in TG narcoleptic mice compared with the WT mice group (17.6 ± 1.6 pg/mL vs 35.8 ± 1.8 pg/mL, p < 0.0001). Thirty of 100 TG mice had undetectably low blood hypocretin levels, whereas only 1 of 96 WT mice had undetectable levels. The specificity of undetectable blood levels in TG mice was high (99%), although the sensitivity was only 30%. (C) Similarly, blood hypocretin levels were significantly lower in KO narcoleptic mice compared with a WT mice group (26.1 ± 3.0 pg/mL vs 36.3 ± 2.6 [SEM] pg/mL, p = 0.015). However, the overlap in hypocretin values between KO and WT mice was larger than that of the TG mice comparison. Four of 23 KO mice and 3 of 21 WT mice showed undetectable blood hypocretin levels.

In these narcoleptic mice, serum hypocretin signals were measured by RIA without extraction using a rabbit antihypocretin-1 polyclonal antibody produced in house with high sensitivity (IC90 = 0.5 pg/mL). We found that blood hypocretin levels were significantly lower in both KO and TG narcoleptic mice compared with their respective wild-type mice groups (Fig. 2). Most strikingly, 30 of 100 TG mice had undetectably low blood hypocretin levels, whereas only 1 of 96 wild-type mice had undetectable levels (21% of TG mice overlapped with the mean ± 2SD of control level) (Fig. 2). The specificity of undetectable blood levels in TG mice was high (99%), although the sensitivity was only 30%. Notably, the overlap in hypocretin values between KO and WT mice (39% of KO mice overlapped, with a mean ± 2SD of the control level) was greater than that of the TG mice.

Therefore, hypocretin signals in the blood (detected with RIA using a polyclonal antibody) reflect hypocretin deficiency status in both hypocretin-deficient narcoleptic mice models (gene vs cell targeting model). Although hypocretin status in peripheral organs has not been investigated in orexin/ataxin-3 narcoleptic mice, no hypocretin production in peripheral organs (and brain) was assumed in KO mice. Thus the overlap observed in hypocretin signals between KO and WT mice is probably caused by large background signals in the blood. By reducing the background signals, e.g., by applying larger sample volumes or extraction methods together with more sensitive and specific assays, we may be able to diagnose narcoleptic mice reliably by blood hypocretin measures. Similarly, if major blood hypocretin signals come from the brain, or if peripheral organ hypocretin is also deficient in human narcolepsy we may be able to develop a blood assay for detecting hypocretin- deficient human narcolepsy.

Several blood preparation factors may also have affected the ability to detect hypocretins in the blood. It needs to be determined whether plasma or serum hypocretin levels and the use of different types of protease inhibitors (aprotinin and protease cocktails) can be more appropriately measured. If we use plasma, the collection method (e.g., in EDTA, anticoagulant citrate dextrose [ACD], or heparin) is an important issue (see ref. 30). The various extraction techniques (organic solvents vs Sep-Pak methods) and purification methods (HPLC and antibody-mediated affinity column) also need to be considered. Finally, the RIA conditions should also be varied, as some peptides are better detected under nonequilibrium conditions.

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