Peripheral insulin administration attenuates the increase in neuropeptide Y concentrations in the hypothalamic arcuate nucleus of fasted rats

Fasting increases neuropeptide Y (NPY) concentrations in the arcuate nucleus (ARC), its site of synthesis, and in other regions of the rat hypothalamus. Neuropeptide Y is a potent central orexigenic agent and may therefore stimulate appetite during fasting. We tested the hypothesis that low plasma insulin levels stimulate ARC levels of NPY in fasted rats. Compared with freely fed controls (n = 8), rats fasted for 72 h (n = 8) showed significantly lower plasma insulin levels (28.9 ± 1.6 vs. 52.6 ± 5.7 pmol/l; p < 0.001) and higher ARC NPY concentrations (14.2 ± 1.8 vs. 8.4 ± 2.2 fmol/μg protein; p < 0.001). Fasted rats treated with subcutaneous insulin (5 U/kg/day; n = 10), which nearly normalized plasma insulin (46.6 ± 2.8 pmol/l), showed intermediate ARC NPY levels (11.2 ± 1.4 fmol/μg protein; p < 0.01 vs. controls and untreated fasted rats). Insulin administered peripherally, therefore, attenuates fasting-induced NPY increases in the ARC, supporting the hypothesis that hypoinsulinemia stimulates hypothalamic NPY.     

An immunoradiometric assay for the measurement of neuropeptide Y in plasma

The development of an immunoradiometric assay (IRMA) for the direct measurement of neuropeptide Y (NPY) concentrations in plasma is reported. The assay employs simultaneous addition of ¹²⁵I-labelled affinity purified sheep anti-(NPY 31–36) immunoglobulin (IgG) and a rabbit anti-NPY serum to 0.25 ml volumes of standard or unknown. After 16 hr incubation at 4°C NPY-bound labelled IgG is precipitated using sheep anti-(rabbit IgG Fc region) IgG coupled to Dynospheres solid phase. Precipitated counts are proportional to the NPY concentration in samples. Using this methodology it is possible to measure basal levels in normal human subjects (range 1–5 fmol/ml). Technical difficulties encountered in raising “site-specific” antisera to NPY during the establishment of this assay are outlined.     

Attenuated feeding responses to circadian and palatability cues in mice lacking neuropeptide Y

Neuropeptide Y (NPY) is a potent orexigenic peptide that is implicated in the feeding response to a variety of stimuli. The current studies employed mice lacking NPY (Npy−/−) and their wild-type (Npy+/+) littermates to investigate the role of this peptide in the feeding response to circadian and palatability cues. To investigate the response to a circadian stimulus, we assessed food intake during the 4-h period following dark onset, a time of day characterized by maximal rates of food consumption. Compared to Npy+/+ controls, intake of Npy−/− mice was reduced by 33% during this period (0.6 ± 0.1 g versus 0.9 ± 0.1 g; p ≤ 0.05). In contrast, intake did not differ between genotypes when measured over a 24-h period (3.7 ± 0.2 g versus 3.5 ± 0.3 g; p = ns). Furthermore, reduced dark cycle 4 h food intake in Npy−/− mice was not evident after a 24-h fast (1.4 ± 0.1 g for both genotypes; p = ns), despite a pronounced delay in the initiation of feeding (636 ± 133 s versus 162 ± 29 s; p ≤ 0.05). To investigate the role of NPY in the feeding response to palatability cues, mice were presented with a highly palatable diet (HP) for 1 h each day (in addition to having ad libitum access to chow) for 18 days. Npy+/+ mice rapidly increased daily HP intake such that by the end of the first week, they derived a substantial fraction of daily energy from this source (41 ± 3%). By comparison, HP intake was markedly reduced in Npy−/− mice during the first week (24 ± 7% of daily energy intake, p ≤ 0.05 versus Npy+/+), although it eventually increased (by Day 9) to values comparable to those of Npy+/+ controls. These experiments suggest that NPY contributes to the mechanism whereby food intake increases in response to circadian and palatability cues and that mechanisms driving food intake in response to these stimuli differ from those activated by energy restriction.     

Localization and identification of Neuropeptide Y (NPY)-like immunoreactivity in the frog brain

The distribution of neuropeptide Y (NPY) in the central nervous system of the frog Rana ridibunda was determined by immunofluorescence using a highly specific antiserum. NPY-like containing perikarya were localized in the infundibulum, mainly in the ventral and dorsal nuclei of the infundibulum, in the preoptic nucleus, in the posterocentral nucleus of the thalamus, in the anteroventral nucleus of the mesencephalic tegmentum, in the part posterior to the torus semicircularis, and in the mesencephalic cerebellar nucleus. Numerous perikarya were also distributed in all cerebral cortex. Important tracts of immunoreactive fibers were found in the infundibulum, in the preoptic area, in the lateral amygdala, in the habenular region, and in the tectum. The cerebral cortex was also densely innervated by NPY-like immunoreactive fibers. A rich network of fibers was observed in the median eminence coursing towards the pituitary stalk. Scattered fibers were found in all other parts of the brain except in the cerebellum, the nucleus isthmi and the torus semicircularis, where no immunoreactivity could be detected. NPY-immunoreactive fibers were observed at all levels of the spinal cord, with particularly distinct plexus around the ependymal canal and in the distal region of the dorsal horn. At the electron microscope level, NPY containing perikarya and fibers were visualized in the ventral nuclei of the infundibulum, using the peroxidase-antiperoxidase and the immunogold techniques. NPY-like material was stored in dense core vesicles of 100 nm in diameter. A sensitive and specific radioimmunoassay was developed. The detection limit of the assay was 20 fmole/tube. The standard curves of synthetic NPY and the dilution curves for acetic acid extracts of cerebral cortex, infundibulum, preoptic region, and mesencephalon plus thalamus were strictly parallel. The NPY concentrations measured in these regions were (pmole/mg proteins) 163±8, 233±16, 151±12 and 60±13, respectively. NPY was not detectable in cerebellar extracts. After Sephadex G-50 gel filtration of acetic acid extracts from whole frog brain, NPY-like immunoreactivity eluted in a single peak. Reverse phase high performance liquid chromatography (HPLC) and radioimmunoassay were used to characterize NPY-like peptides in the frog brain. HPLC analysis revealed that infundibulum, preoptic area and telencephalon extracts contained a major peptide bearing NPY-like immunoreactivity. The retention times of frog NPY and synthetic porcine NPY were markedly different. HPLC analysis revealed also the existence, in brain extracts, of several other minor components cross-reacting with NPY antibodies. These results provide the first evidence for the presence of NPY in the brain of a non-mammalian chordate and indicate that the structure of NPY is preserved among the vertebrate phylum. The abundance of NPY producing neurons in the hypothalamus and telencephalon suggests that this peptide may play both neuroendocrine and neurotransmitter functions in amphibians.     

Neuropeptide Y receptor agonists: Multiple effects on spontaneous activity in the paraventricular hypothalamus

In vitro rat hypothalamic slices were used to examine the ability of neuropeptide Y (NPY), and the putative Y₁ and Y₂ receptor agonists [Pro³⁴]NPY, and [C²]NPY, to modify spontaneous single-neuron discharge in the paraventricular nucleus (PVN). NPY and [Pro³⁴]NPY, at high concentrations (1500 nM), decreased discharge rates. At intermediate concentrations (150 nM) these peptides produced multiple effects, including increases, decreases, and biphasic changes. At lower concentrations (0.15–15 nM), they typically increased discharge rates. In contrast, [C²]NPY, at all concentrations (1.5–1500 nM), predominantly increased discharge rates. Thus, these NPY sybtype agonists have multiple effects on discharge rate, which may be due to action on multiple NPY receptor subtypes.     

Evaluation of heat shock protein 70 as a biomarker of environmental stress in Fucus serratus and Lemna minor

Heat shock proteins (Hsps) are known to be induced in response to short-term stress. The present study aimed to evaluate the potential of Hsp70 as a biomarker of stress produced by increased temperature, osmotic pressure, and exposure to cadmium and sodium chloride in marine macroalgae and fresh water plant species. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was developed with a working range of 0.025-10 μg ml^-1 using a monoclonal antibody raised against purified Hsp70 of Phaseolus aureus (mung bean). Fucus serratus (toothed wrack), Chondrus crispus (Stackhouse or Carrageen moss), Ulva lactuca (sea lettuce) and Lemna minor (common duckweed) sample extracts were stressed for up to 24 h and then tested in the IC-ELISA. The presence of Hsp70 and cross-reactivity of the monoclonal antibody was confirmed by Western blot. The heat shock response was confirmed in each species using a 2-h 42°C treatment. Following heat shock, Hsp70 concentrations increased to a peak at 2 h (F. serratus) or 4 h (L. minor), after which concentrations decreased. Osmotic and cadmium stresses also resulted in elevated Hsp70 concentrations in samples of F. serratus and L. minor when compared with unstressed controls. In both, osmotic and metal stress, the production of Hsp70 increased to a maximum and subsequently decreased as the stressor levels increased. Results suggest that Hsp70 IC-ELISA could potentially be applied to the detection of stress in these aquatic species, although it would probably be most effective when used in conjunction with other measurements to provide a stressor-specific biomarker profile or fingerprint.     

Homogeneous amperometric immunoassay for theophylline in whole blood

A homogeneous spectrophotometric EMIT immunoassay kit for the quantitation of theophylline in serum or plasma has been modified to produce a rapid, amperometric immunoassay requiring a 50 μl whole blood sample. The basis of the detection system for the assay is the electrochemical oxidation of NADH produced by G6PDH-labelled theophylline at a potential of + 150 mV vs Ag/AgCl using platinised activated carbon (PACE) electrodes. Comparison of the amperometric whole blood method with the conventional spectrophotometric plasma assay produced a reasonable correlation: Y = 0·90x − 1·01, (r = 0·98, N = 12). The advantage of the new method is that simple and robust instrumentation can rapidly determine theophylline in whole blood with no sample pre-treatment or separation steps.     

High-performance liquid chromatographic assay for the measurement of the novel microtubule inhibitor 1069C85 in biological tissues and fluids

1069C85 is a novel tubulin binder developed to circumvent the resistance associated with the Vinca alkaloids. Cytotoxic activity has been demonstrated in vitro against a variety of tumour cell lines, including a variant of the P388 leukaemia with acquired resistance to vincristine. A phase I clinical trial is planned and an assay suitable for preclinical and clinical pharmacokinetics has been developed. A high-performance liquid chromatographic (HPLC) assay is described which allows measurement of 1069C85 in plasma, urine, and tissue samples. The method uses reversed-phase chromatography with isocratic elution and detection by fluorescence at 406 nm following excitation at 340 nm. The assay is specific, sensitive (limit of sensitivity 0.25 ng/ml) and reproducible (coefficient of variation <5%). The method has been used to study the pharmacokinetics of 1069C85 in Balb C mice following a single oral dose of 1 mg/kg. The maximum plasma concentration was reached 15 min after administration and subsequent elimination was slow with a half life of 6.5 ± 2.2 h. The drug remained detectable in plasma, at 1 ± 0.5 ng/ml, 24 h after this dose. This assay will be used to determine the pharmacokinetic profile of 1069C85 in mice and in a forthcoming phase I clinical trial.     

Neuropeptide Y: Direct and indirect action on insulin secretion in the rat

Neuropeptide Y (NPY) was tested for an ability to directly influence the release of insulin using an in vitro isolated rat pancreatic islet system. NPY, at doses ranging from 100 pg/ml to 1 μg/ml, had no significant effect on the basal release (5.5 mM glucose) of insulin. However, NPY treatment resulted in a significant, dose-dependent (1 ng/ml to 1 μg/ml) inhibition of glucose-stimulated (11 mM) insulin release. When tested in a perfused rat pancreas preparation in situ, NPY administration led to a marked inhibition of both basal and stimulated insulin release followed by a postinhibitory rebound which exceeded the control insulin levels by 3-fold. In contrast, the intracerebroventricular (ICV) microinjection of NPY (5 μg) produced a significant but delayed (30 min) elevation of circulating insulin. It is therefore suggested that the direct action of NPY on insulin release is inhibitory while the central action of NPY indirectly results in an increase in plasma insulin. Thus, NPY may be added to the growing list of peptidergic agents which may affect the endocrine pancreas by acting as neurotransmitters and/or neuromodulators.     

Diel thermoregulation of the crawfish Procambarus Clarkii (crustacea, cambaridae)

1. 1. In a diel cycle Procambarus clarkii has two preferred temperatures: 24.0 ± 0.15 SEM °C during the day and 26.7 ± 0.13 SEM °C at night.

2. 2. The preferred temperatures are independent from the weight of the organisms.

3. 3. In the photophase the animals are dispersed, in the scotophase they congregate.

4. 4. The crawfish seem to feed during the thermal interphases.

5. 5. Animals in a constantly dark condition maintain a diel preferendum of temperature.

Selective determination of sultopride in human plasma using high-performance liquid chromatography with ultraviolet detection and particle beam mass spectrometry

We developed a sensitive and selective method for determining levels of sultopride, a neuroleptic drug of the substituted benzamide, in human plasma using high-performance liquid chromatography (HPLC) combined with UV detection and particle beam mass spectrometry (PBMS). Sutopride was extracted with tert.-butylmethyl ether using a salting-out technique. Tiapride served as an internal standard (I.S.). Sutopride and I.S. were separated by HPLC on a silica column with a mobile phase of acetonitrile-0.1 M ammonium acetate (94:6, v/v). The calibration curves were linear over the concentration range from 5 to 1000 ng/ml by HPLC with UV detection and from 10 to 1000 ng/ml with PBMS detection. The limit of quantitation was 5 ng/ml with UV detection and 10 ng/ml with PBMS detection. The absolute recovery was 92% and the within-day coefficients of variation were 2.9–7.1% at plasma concentrations from 50 to 500 ng/ml, determined by HPLC with UV detection. Using this method, we measured the plasma concentrations of sultopride with replicate analyses in four hospitalized patients and steady-state plasma levels were determined to be 161.6±30.8, 321.1±93.7, 726.5±143.1 and 1273.6±211.2 ng/ml, respectively.     

Chromatographic characterisation of the circulating neuropeptide Y immunoreactivity from patients with phaeochromocytoma

Samples of adrenal medullary phaeochromocytoma from two patients were extracted in acid. The tissue concentrations of neuropeptide Y (NPY)-like immunoreactivity were found to be 0.94 and 8.11 micrograms/g. Plasma from these patients subjected to NPY radioimmunoassay after Sephadex G-75 gel filtration chromatography revealed circulating plasma concentrations of 1889 and 2079 pg/ml, compared to levels in normal plasma of less than 50 pg/ml, treated in the same way. Further characterisation of this plasma NPY-like immunoreactivity by HPLC showed this peptide to possess identical elution behaviour to human NPY standard. It is suggested that circulating NPY may be a contributory factor in the symptoms exhibited by some patients with adrenal medullary phaeochromocytoma.     

Plasma and scales levels of interleukin 18 in comparison with other possible clinical and laboratory biomarkers of psoriasis activity

The aim of the study was to assess plasma and scales levels of interleukin (IL) 18 collected from psoriatic patients with different disease activity. IL-18 concentrations were measured using an enzyme immunoassay in the plasma and scales of 34 patients with chronic plaque type psoriasis. IL-18 levels were analysed with respect to plasma-transforming growth factor β₁ (TGF-β₁), the disease duration and the duration of the present relapse, and psoriasis area and severity index (PASI). Plasma IL-18 concentration varied from 90 to 1300 pg ml^-1 and means (368.2±42.4 pg ml^-1) were significantly elevated in comparison with healthy controls (205.9±31.8 pg ml^-1). The presence of IL-18 was also demonstrated in scales from skin lesions. Treatment caused a significant decrease of plasma IL-18 concentration to 250.2±13.8 pg ml^-1. There was a significant correlation between plasma IL-18 levels and PASI values (r=0.554). There was no correlation between IL-18 concentration in scales and PASI, between IL-18 concentrations in plasma and scales, and between plasma IL-18 and the disease duration or duration of present relapse. Plasma TGF-β₁ concentration demonstrated a significant correlation with PASI (r=0.353), but not with IL-18 levels in plasma (r=0.063) and scales (0.141). The sum of plasma levels of IL-18 and TGF-β₁ divided by the optimal coefficient demonstrated a statistically significant correlation with the highest r-value. The findings confirm an association between plasma IL-18 concentration and psoriasis severity. Moreover, it was shown that combined measurement of IL-18 and TGF-β₁ in plasma can be considered as a possible biomarker of psoriasis activity.     

Plasma copper and lipid peroxidation in cigarette smokers

Plasma levels of copper and lipid peroxidation were evaluated in 14 smokers as compared to 14 nonsmokers. Plasma copper concentrations were higher in smokers than in nonsmokers (122.5 ± 19.15 vs. 101.5 ± 16.2 μg/dl, P < .01). Plasma lipoperoxidation, evaluated as fluorescent damage products of lipid peroxidation (FDPL), also was higher in smokers than in nonsmokers (20.35 ± 2.6 vs. 17.1 ± 2.95 units of relative fluorescence/ml, P < .01). A significant and positive correlation between the number of cigarettes smoked, expressed as pack years, and the levels of either FDPL (r = .61, P < .025) or copper (r = .55, P < .05) was found. Moreover, a significant and positive relationship between copper and FDPL values was observed in smokers (r = .64; P < .025), but not in nonsmokers. These data indicate that cigarette smoke-related plasma oxidant load may be partly due to enhanced levels of the prooxidant metal copper, potentially suggesting the supplementation of specific antioxidants (e.g., zinc) to counteract cigarette smoke-induced oxidative stress in smokers.     

Does temperature preference relate to the anaerobic capacity of Atlantic cod (Gadus morhua L.) with different haemoglobin phenotype?

The effect of Hb-I* phenotype on white muscle lactate dehydrogenease (LDH, E. C. 1.1.1.27) activity and buffering capacity was studied in Atlantic cod (Gadus morhua), acclimated and measured at temperatures near their behavioral temperature preference. It was hypothesized that these conditions would optimize biochemical processes but no difference was found in LDH activity between the Hb-I* phenotype after 56 d of acclimation to 6 and 14°C. However, LDH activity was both mass- and temperature-dependent; mean activity was 162.2±5.0 and 275.9±6.4 IU g^-1 wet mass (mean±SEM) at 6 and 14°C respectively and larger fish had the highest rate of enzyme activity. White muscle buffer capacity was unaffected by Hb-I* phenotype but higher in cod held at 14°C.     

Characterization of growth hormone-releasing hormone (GHRH) binding to cloned porcine GHRH receptor

To study structure-activity relationships of growth hormone-releasing hormone (GHRH), a competitive binding assay was developed using cloned porcine adenopituitary GHRH receptors expressed in human kidney 293 cells. Specific binding of [His¹,¹²⁵I-Tyr¹⁰,Nle²⁷]hGHRH(1–32)-NH₂ increased linearly with protein concentration (10–45 μg protein/tube). Binding reached equilibrium after 90 min at 30°C and remained constant for at least 240 min. Binding was reversible to one class of high-affinity sites (K_d = 1.04 ± 0.19 nM, B_max = 3.9 ± 0.53 pmol/mg protein). Binding was selective with a rank order of affinity (IC₅₀) for porcine GHRH (2.8 ± 0.51 nM), rat GHRH (3.1 ± 0.69 nM), [N-Ac-Tyr¹, -Arg²]hGHRH(3–29)-NH₂ (3.9 ± 0.58 nM), and [ -Thr⁷]GHRH(1–29)-NH₂ (189.7 ± 14.3 nM), consistent with their binding to a GHRH receptor. Nonhydrolyzable guanine nucleotides inhibited binding. These data describe a selective and reliable method for a competitive GHRH binding assay that for the first time utilizes rapid filtration to terminate the binding assay.     

Release of neuropeptide Y and hemodynamic changes during surgical removal of human pheochromocytomas

This study investigates the release of Neuropeptide Y from eight human pheochromocytomas. Profil immunoreactive Neuropeptide Y (Ir-NPY) levels during the management of surgery were compared with these of norepinephrine (NE) while hemodynamics were monitored. Plasma IrNPY and NE levels increased during tumor manipulation and returned to near normal one hour after operation. However, Ir-NPY levels remained high just after tumor resection while NE levels were significantly decreased. At tumor manipulation and just after tumor resection, plasma Ir-NPY levels were correlated with the systemic vascular resistances (SVR) (r = 0.74; P<0.04 and r = 0.86; P<0.006 respectively). No correlation was found either between plasma Ir-NPY and NE levels or between plasma NE levels and SVR. The release of Ir-NPY from tumor tissue, studied by a superfusion method, exhibited a significant correlation with the plasma Ir-NPY concentrations at the time of corresponding tumor resection (r = 0.95; P<0.007). Chromatographic analysis showed that Ir-NPY in plasma and outflow migrate as human NPY (1-36). These results confirmed that in pheochromocytoma, plasma NPY mainly originates from the tumor and argue for an important role of NPY in pheochromocytoma hypertension as indicated by the correlation between the Ir-NPY levels and the SVR.     

Sensitive high-performance liquid chromatographic determination of indomethacin in human plasma: Pharmacokinetic studies after single oral dose

A sensitive high-performance liquid chromatographic assay for the specific determination of indomethacin at concentrations down to 20 ng/ml in human plasma is described.

This method has been applied to investigate the disappearance of indomethacin from plasma of ten subjects following the intake of two formulations (Indocid® and generic form). An initial half-life of 1.32 ± 0.44 h⁻¹ was found which is in good agreement with other findings, but the terminal phase was much longer (13.6 ± 6.9 h⁻¹) than previously reported. There is no difference between the two galenic forms (p <0.001).     

Impaired resistance to oxidation of low density lipoprotein in cystic fibrosis: Improvement during vitamin E supplementation

Antioxidants such as vitamin E protect unsaturated fatty acids of LDL against oxidation. In the ex vivo model used, LDL was exposed to Cu²⁺ ions, a potent prooxidant capable of initiating the oxidation of LDL. The lag time, indicating the delay of conjugated diene formation in LDL due to antioxidant protection, was measured in 54 cystic fibrosis (CF) patients with plasma -tocopherol levels below (Group A, n = 30) or above (Group B, n = 24) 15.9 μmol/L (mean - 2 SD of Swiss population). Patients were reevaluated after 2 months on 400 IU/d of oral RRR--tocopherol. In group A, -tocopherol concentrations in LDL increased significantly from 3.2 ± 1.6 mol/mol LDL to 8.2 ± 2.8 mol/mol (P < 0.001) and lag times increased from 79 ± 33, min to 126 ± 48 min (P < 0.001), whereas in the vitamin E sufficient group B no further increase neither in LDL -tocopherol concentrations or in lag times was observed. LDL oleic acid concentrations were higher, and linoleic acid concentrations were lower in patients than in controls. After efficient vitamin E supplementation, lag times were positively related to LDL -tocopherol (P < 0.01) and negatively to LDL linoleic and arachidonic acid content (P < 0.001). The maximum rate of oxidation correlated positively with linoleic and arachidonic acid concentrations, as did the maximum conjugated diene absorbance. These results indicate that LDL resistance to oxidation is impaired in vitamin E deficient CF patients but can be normalized within 2 months when -tocopherol is given in sufficient amounts. Linoleic and arachidonic acid content exhibit a major influence on the LDL resistance to oxidation.     

Effects of quantitative and qualitative variation in phenolic compounds on feeding in three species of marine invertebrate herbivores

The deterrent effects of brown algal phenolic compounds and the terrestrial polyphenolic tannic acid on feeding by three species of invertebrate herbivores from central California, including the gastropods Tegula funebralis (Adams) and Tegula brunnea (Phillipi) and the echinoid Strongylocentrotus purpuratus (Stimpson) were examined. Algal phenolics used were the monomeric phenolic phloroglucinol and polyphloroglucinols from Fucus vesiculosus (Linnaeus), Halidrys siliquosa (Linnaeus) Lyngbye and Eisenia arborea Areschoug. All of the polyphenolics deterred feeding by all three herbivores at concentrations of 5 mg · ml⁻¹ in agar disks. Concentrations of 2 mg · ml⁻¹ also generally deterred feeding by the gastropods (these levels were not tested against S. purpuratus). Relative amounts of deterrence by different compounds were similar, especially for the gastropods. Phloroglucinol deterred feeding by the echinoids, but not by T. funebralis. Responses of the echinoids were otherwise similar to the gastropods, but more variable. I also demonstrated deterrence of S. purpuratus by tannic acid using the “tanned” kelp technique of Steinberg (1985). Reactivity of the different phenolic compounds in the Folin-Denis procedure, a common colorimetric assay used to estimate levels of phenolics in plant tissue, was similar. This suggests that measuring phenolic levels in brown algae by this technique will not be greatly confounded by the occurrence of different kinds of phenolic molecules in different brown algae. This result, in combination with the similarity of the deterrent effects of the compounds used in this study, increases the validity of previous studies in the northeastern Pacific Ocean which correlate algal phenolic levels and diets or feeding preferences of invertebrate herbivores. For plants and herbivores in this region, this assay is a reasonable measure of a biologically meaningful phenomenon — levels of phenolic deterrents in the algae.