In-Cell Western analysis of Helicobacter pylori-induced phosphorylation of extracellular-signal related kinase via the transactivation of the epidermal growth factor receptor

Helicobacter pylori activates extracellular-signal related (ERK) kinases in gastric epithelial cells, via transactivation of the EGF receptor (EGFR). H. pylori activation of EGFR may be relevant to epithelial hyperproliferation and gastric carcinogenesis. The aim of this study was to develop an 'In-Cell Western' (ICW) assay for quantitative examination of H. pylori-induced epithelial signalling, to enable the role of the EGFR in H. pylori-induced phosphorylation of ERK in epithelial cells to be ascertained. H. pylori strains were co-incubated with A431 and AGS cells. pERK and total ERK were quantified in situ using ICW analysis. H. pylori strains both with, and without a cag PAI, and Helicobacter felis, significantly increased pERK levels in A431 cells. The EGFR inhibitor EKB-569 dose-dependently reduced H. pylori-induced ERK phosphorylation in A431 and AGS cells. A significantly lower reduction was observed with cag+ strains in A431 but not AGS cells. The cag PAI was not necessary for EGFR signal transactivation. These data suggest that H. pylori induces pERK in epithelial cells partly via the EGFR pathway. Additional signalling mechanisms are likely to be involved in H. pylori-induced ERK phosphorylation. ICW analysis is a rapid quantitative method for evaluating the effects of inhibitors on H. pylori-induced cell signalling pathways of relevance to gastric carcinogenesis.     

The predominant mechanism of intercellular calcium wave propagation changes during long-term culture of human osteoblast-like cells

Intercellular calcium waves (ICW) are calcium transients that spread from cell to cell in response to different stimuli. We previously demonstrated that human osteoblast-like cells in culture propagate ICW in response to mechanical stimulation by two mechanisms. One mechanism involves autocrine activation of P2Y receptors, and the other requires gap junctional communication. In the current work we ask whether long-term culture of osteoblast-like cells affects the propagation of ICW by these two mechanisms. Human osteoblast-like cells were isolated from bone marrow. Mechanically induced ICW were assessed by video imaging of Fura-2 loaded cells after 1, 2 and 4 months culture. The P2Y2 receptor and the gap junction protein Cx43 were assessed by Western blot and real-time PCR. In resting conditions, P2Y mediated ICW prevailed and spread rapidly to about 13 cells. P2Y receptor desensitization by ATP disclosed gap junction-mediated ICW which diffused more slowly and involved not more than five to six cells. After 2 months in culture, ICW appeared slower and wave propagation was much less inhibited by P2Y desensitization, suggesting an increase in gap junction-mediated ICW. After 4 months in culture cells still responded to addition of ATP, but P2Y desensitization did not inhibit ICW propagation. Our data indicate that the relative role of P2Y-mediated and gap junction-mediated ICW changes during osteoblast differentiation in vitro. In less differentiated cells, P2Y-mediated ICW predominate, but as cells differentiate in culture, gap-junction-mediated ICW become more prominent. These results suggest that P2Y receptor-mediated and gap junction-mediated mechanisms of intercellular calcium signaling may play different roles during differentiation of bone-forming cells.     

Quantification of Rapid Myosin Regulatory Light Chain Phosphorylation Using High-Throughput In-Cell Western Assays: Comparison to Western Immunoblots

Background

Quantification of phospho-proteins (PPs) is crucial when studying cellular signaling pathways. Western immunoblotting (WB) is commonly used for the measurement of relative levels of signaling intermediates in experimental samples. However, WB is in general a labour-intensive and low-throughput technique. Because of variability in protein yield and phospho-signal preservation during protein harvesting, and potential loss of antigen during protein transfer, WB provides only semi-quantitative data. By comparison, the “in-cell western” (ICW) technique has high-throughput capacity and requires less extensive sample preparation. Thus, we compared the ICW technique to WB for measuring phosphorylated myosin regulatory light chain (PMLC₂₀) in primary cultures of uterine myocytes to assess their relative specificity, sensitivity, precision, and quantification of biologically relevant responses.

Methodology/Principal Findings

ICWs are cell-based microplate assays for quantification of protein targets in their cellular context. ICWs utilize a two-channel infrared (IR) scanner (Odyssey®) to quantify signals arising from near-infrared (NIR) fluorophores conjugated to secondary antibodies. One channel is dedicated to measuring the protein of interest and the second is used for data normalization of the signal in each well of the microplate. Using uterine myocytes, we assessed oxytocin (OT)-stimulated MLC₂₀ phosphorylation measured by ICW and WB, both using NIR fluorescence. ICW and WB data were comparable regarding signal linearity, signal specificity, and time course of phosphorylation response to OT.

Conclusion/Significance

ICW and WB yield comparable biological data. The advantages of ICW over WB are its high-throughput capacity, improved precision, and reduced sample preparation requirements. ICW might provide better sensitivity and precision with low-quantity samples or for protocols requiring large numbers of samples. These features make the ICW technique an excellent tool for the study of phosphorylation endpoints. However, the drawbacks of ICW include the need for a cell culture format and the lack of utility where protein purification, concentration or stoichiometric analyses are required.     

Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.     

Assay development and high-throughput screening of small molecular c-Abl kinase activators

A 2-step kinase assay was developed and used in a high-throughput screen (HTS) of more than 1 million compounds in an effort to identify c-Abl tyrosine kinase activators. This assay employed a 2-step phosphorylation reaction: in the first step, purified recombinant c-Abl was activated by incubating with compound in the presence of adenosine triphosphate (ATP). In the second step, the TAMRA-labeled IMAP Abltide substrate was added to allow phosphorylation of the substrate to occur. The assay was calibrated such that inactive c-Abl protein was activated by ATP alone to a degree that it not only demonstrated a measurable c-Abl activity but also maintained a robust assay window for screening. The screen resulted in 8624 primary hits with >30% response. Further analysis showed that 1024 had EC(50) <10 μM with a max % response of >50%. These hits were structurally and chemically diverse with possibly different mechanisms for activating c-Abl. In addition, selective hits were shown to be cell permeable and were able to induce c-Abl activation as determined by In-Cell Western (ICW) analysis of HEK-MSRII cells transduced with BacMam virus expressing full-length c-Abl.     

Measurement of nutritional status in simulated microgravity by bioelectrical impedance spectroscopy.

The potential of bioelectrical impedance spectroscopy (BIS) for assessing nutritional status in spaceflight was tested in two head-down-tilt bed-rest studies. BIS-predicted extracellular water (ECW), intracellular water (ICW), and total body water (TBW) measured using knee-elbow electrode placement were compared with deuterium and bromide dilution (DIL) volumes in healthy, 19- to 45-yr-old subjects. BIS was accurate during 44 h of head-down tilt with mean differences (BIS - DIL) of 0-0.1 kg for ECW, 0.3-0.5 for ICW, and 0.4-0.6 kg for TBW (n = 28). At 44 h, BIS followed the within-individual change in body water compartments with a relative prediction error (standard error of the estimate/baseline volume) of 2.0-3.6% of water space. In the second study, BIS did not detect an acute decrease (-1.41 +/- 0.91 kg) in ICW secondary to 48 h of a protein-free, 800 kcal/day diet (n = 18). BIS's insensitivity to ICW losses may be because they were predominantly (65%) localized to the trunk and/or because there was a general failure of BIS to measure ICW independently of ECW and TBW. BIS may have potential for measuring nutritional status during spaceflight, but its limitations in precision and insensitivity to acute ICW changes warrant further validation studies.     

Changes in glycine and leucine transport during red cell maturation in the rat

Both glycine and leucine transport in rat red blood cells have been studied. The glycine uptake showed two different components, one sodium-dependent and another diffusion-like process. In contrast, leucine uptake was sodium independent. Both, Na⁺-dependent glycine and the overall leucine uptake in red blood cells showed a saturable pattern. Kinetic parameters in reticulocytes were: i) glycine: apparent Km 0.16 mM; V_max 100.2 nmol/ml ICW/min; ii) leucine: apparent Km 2.11 mM; V_max 3.88 mol/ml ICW/min. The erythrocytes kinetic parameters were: i) glycine: apparent Km 0.17 mM; V_max 9.47 nmol/ml ICW/min; leucine; apparent Km 4.77 mM; V_max 7.42 mol/ml ICW/min. The Kd values (sodium independent glycine uptake) were similar in both kind of cells, but the importance of this component in total glycine uptake in erythrocytes was much higher than in reticulocytes. Our results confirm that rat red blood cells have both saturable leucine and Na⁺-dependent glycine uptake, but some important changes occur during cell maturation.     

Relationship between changes in total-body water and fluid distribution with maximal forearm strength in elite judo athletes

Among judo athletes, strong grip strength is crucial for performing offensive and defensive maneuvers that rely predominantly on forearm maximal strength (FMS). The study aims were to evaluate changes in total-body water (TBW) and its compartments (extracellular water [ECW] and intracellular water [ICW]) and their relationship with loss of FMS in elite judo athletes. At baseline (weight stability), 27 male elite athletes were evaluated (age: 23.2 ± 2.8 years) and again evaluated 1-3 days before competition. Athletes were free to gain or lose weight based upon their specific competition needs. Using dilution techniques (deuterium and bromide), TBW and ECW were estimated, and ICW was calculated (ICW = TBW - ECW). Fat, fat-free mass, and appendicular lean soft tissue (LST) were assessed by dual-energy x-ray absorptiometry. Handgrip was used to assess FMS. Using a reduction of 2% as a representative outcome for decreased FMS, 10 athletes were identified as having lost FMS, whereas 17 changed <2% or gained. Comparison of means and logistic regression analysis were performed. Results from baseline to before competition indicated that those who lost ≥2% of FMS significantly decreased TBW and ICW by -2.7 ± 3.0 and -4.4 ± 4.2%, respectively. The groups differed in ICW changes (-4.4 ± 4.2 vs. 1.9 ± 6.1%), respectively, for those who lost FMS by ≥2%. The ICW changes, but not in TBW or ECW, significantly predicted the risk of losing FMS (β = 0.206; p = 0.027), even adjusting for weight and arm LST changes. These findings indicated that reductions in ICW increased the risk of losing grip strength in elite judo athletes.     

Electrostatic guarding of bookshelves for mould-free preservation of valuable library books

Old books are highly susceptible to mould infection, and an effective method for avoiding moulding is needed to safely preserve valuable books in library stack rooms. Guarding a bookshelf with an electric field screen is a physical method that prevents airborne spores from entering the space used for book preservation. In this study, insulated conductor wires (ICWs) were used as electrodes to form electric fields. The ICWs were arrayed in parallel and linked to each other and to a direct current voltage generator. The electric field screen consisted of two layers of ICWs, which were negatively and positively charged with equal voltages to make dipoles, ICW(?) and ICW(+). Both ICWs generated an attractive force that captured airborne spores of Penicillium digitatum that were blown inside the screen. The attractive force was directly proportional to the applied voltage. At ≧0.9 kV, the screen exerted sufficient force to capture all airflow-carried spores, but a few spores that were once captured were repulsed out of the electric field when subsequent spores were attracted to positions proximal to them. This phenomenon was explained by creeping discharge between spores located close to each other on the ICW surface. This spore-repulsion problem was resolved by adding an additional ICW layer to the electric field screen, namely an electric field screen with an ICW(?) layer on both sides of an ICW(+) layer. The present study demonstrated that the three-layered electric field screen remained mould-free inside a screen-guarded bookshelf, irrespective of continuous spore exposure.     

Evaluation of bioimpedance spectroscopy for measurements of body water distribution in healthy women before, during, and after pregnancy.

Bioimpedance spectroscopy (BIS) is a technique of interest in the study of human pregnancy because it can assess extracellular (ECW), intracellular (ICW), and total body water (TBW) as ECW plus ICW. The technique requires appropriate resistivity coefficients and has not been sufficiently evaluated during the reproductive cycle. Therefore, in a methodological study, we estimated ECW, ICW, and TBW, by means of BIS, and compared the results with the corresponding estimates obtained by using reference methods. Furthermore, results obtained by means of population-specific resistivity coefficients were compared with results obtained by means of general resistivity coefficients. These comparisons were made before pregnancy, in gestational weeks 14 and 32, as well as 2 wk postpartum in 21 healthy women. The reference methods were isotope and bromide dilution. Average ICW, ECW, and TBW, estimated by means of BIS, were in agreement with reference data before pregnancy, in gestational week 14, and postpartum. The corresponding comparison in gestational week 32 showed good agreement for ICW, whereas estimates by means of BIS were significantly (P < 0.001) lower than the corresponding reference values for ECW and TBW. Thus the BIS technique, which was based on a model developed for the nonpregnant body, estimated increases in ICW accurately, whereas increases in ECW and TBW tended to be underestimated. Estimates obtained by using population-specific and general resistivity coefficients were very similar. In conclusion, the results indicated that BIS is potentially useful for studies during pregnancy but that further work is needed before it can be generally applied in such studies.     

Monitoring of environmental arsenic by cultures of the photosynthetic bacterial sensor illuminated with a near-infrared light emitting diode array

Recombinant Rhodopseudomonas palustris, harboring the carotenoid-metabolizing gene crtI (CrtIBS), and whose color changes from greenish yellow to red in response to inorganic As(III), was cultured in transparent microplate wells illuminated with a light emitting diode (LED) array. The cells were seen to grow better under near-infrared light, when compared with cells illuminated with blue or green LEDs. The absorbance ratio of 525 to 425 nm after cultivation for 24 h, which reflects red carotenoid accumulation, increased with an increase in As(III) concentrations. The detection limit of cultures illuminated with near-infrared LED was 5 microgram/l, which was equivalent to that of cultures in test tubes illuminated with an incandescent lamp. A near-infrared LED array, in combination with a microplate, enabled the simultaneous handling of multiple cultures, including CrtIBS and a control strain, for normalization by the illumination of those with equal photon flux densities. Thus, the introduction of a near-infrared LED array to the assay is advantageous for the monitoring of arsenic in natural water samples that may contain a number of unknown factors and, therefore, need normalization of the reporter event.     

A stochastic two-dimensional model of intercellular Ca2+ wave spread in glia

We describe a two-dimensional stochastic model of intercellular Ca(2+) wave (ICW) spread in glia that includes contributions of external stimuli, ionotropic and metabotropic P2 receptors, exo- and ecto-nucleotidases, second messengers, and gap junctions. In this model, an initial stimulus evokes ATP and UTP release from a single cell. Agonists diffuse and are degraded both in bulk solution and at cell surfaces. Ca(2+) elevation in individual cells is determined by bound agonist concentrations s and by number and features of P2 receptors summed with that generated by IP(3) diffusing through gap junction channels. Variability of ICWs is provided by randomly distributing a predetermined density of cells in a rectangular grid and by randomly selecting within intervals values characterizing the extracellular compartment, individual cells, and interconnections with neighboring cells. Variability intervals were obtained from experiments on astrocytoma cells transfected to express individual P2 receptors and/or the gap junction protein connexin43. The simulation program (available as Supplementary Material) permits individual alteration of ICW components, allowing comparison of simulations with data from cells expressing connexin43 and/or various P2 receptor subtypes. Such modeling is expected to be useful for testing phenomenological hypotheses and in understanding consequences of alteration of system components under experimental or pathological conditions.     

Increased extracellular water compartment, relative to intracellular water compartment, after weight reduction.

The hydration of fat free mass (FFM) and extracellular (ECW) and intracellular water (ICW) compartments were studied in 30 obese premenopausal women before and after a 3-mo weight-reduction program and again after a 9-mo weight-maintenance program. Body fat was determined by a four-compartment model. Total body water and ECW were determined by deuterium dilution and bromide dilution, respectively. After the weight-reduction period, mean weight loss was 12.8 kg, and body fat was reduced on average by 10.9 kg. During weight maintenance, changes in body mass and body fat were not significant. Before weight reduction, mean ECW/ICW ratio was relatively high (0.78 +/- 0.10). During the the study, total body water and ICW did not change significantly. ECW did not change significantly after weight reduction, but 12 mo after the start ECW was significantly increased by 1 liter. The ECW/ICW ratio increased to 0.87 +/- 0.12 (month 12). The hydration of the FFM increased from 74 +/- 1 to 77 +/- 2% during the weight reduction and remained elevated during weight maintenance. In conclusion, the ECW/ICW ratio and the hydration of the FFM, did not normalize during weight reduction and weight maintenance.     

Changes in alanine and glutamine transport during rat red blood cell maturation

Alanine and glutamine transport have been studied during red blood cell maturation in the rat. Kinetic parameters of Na⁺-dependent L-alanine transport were:K _m 0.43 and 1.88 mM andV _max 158 and 45 nmoles/ml ICW/min for reticulocytes and erythrocytes, respectively. During red cell maturation in the rat there is a loss of capacity and affinity of the system ASC for L-alanine transport. The values for Na⁺-dependent L-glutamine transport in reticulocytes wereK _m 0.51 mM andV _max 157 nmoles/ml ICW/min. On the other hand, a total loss of L-glutamine transport mediated by both N and ASC systems is demonstrated in mature red cells. This seems to indicate that during rat red cell maturation the system N disappears. Furthermore, the system ASC specificity in mature cells changes, and glutamine enters the red cell by non-mediated diffusion processes.     

Application of the self-organizing map as a prediction tool for an integrated constructed wetland agroecosystem treating agricultural runoff

A self-organizing map (SOM) model was applied as a prediction tool for the performance of an integrated constructed wetland (ICW) agroecosystem treating agricultural runoff to protect receiving watercourses. By utilizing the SOM model, the time-consuming to measure expensive biochemical oxygen demand outflow concentrations were predicted well by other inexpensive variables, which were quicker and easier to measure. Correct predictions for the outflow biochemical oxygen demand concentrations were between 89% and 100%. This novel approach allows for the real time control of the outflow water quality of the ICW and potentially also of other treatment system applications. Moreover, the missing values and outliers from the large but incomplete ICW data set were replaced accurately by most likely values determined by the SOM model. This was important because the proportions of unusable entries for chemical oxygen demand, suspended solids and biochemical oxygen demand were very high: 41%, 54%, and 61%, respectively.     

Changes in masculine sexual behavior, corticosterone and testosterone in response to acute and chronic stress in male rats

Chronic exposure to stressors increases HPA axis activity and concomitantly reduces HPG axis activity. This antagonistic relationship between both these axes has been proposed to underlie the inhibition of reproductive function due to stress. Sexual behavior in males may be the most vulnerable aspect of male reproduction to acute and chronic stress and it has been suggested that alterations in sexual behavior during stress are due to the antagonistic relationship between testosterone and corticosteroids. However, only in a few studies has a correlation between the levels of testosterone and corticosterone, and sexual behavior been made. In this study, we evaluated the effects of different stressors, applied both acute and chronically, on masculine sexual behavior and whether or not these effects on sexual behavior are accompanied by changes in plasma levels of corticosterone and testosterone. Additionally, we evaluated the effect of testosterone treatment on the effects of stress on sexual behavior. Sexually experienced male rats were exposed to one of the following stressors: immobilization (IMB), electric foot shocks (EFS) or immersion in cold water (ICW). Sexual behavior and plasma levels of testosterone and corticosterone were assessed on days 1, 5, 10, 15, and 20 of stress. In a second experiment, males were castrated, treated with 3 different doses of testosterone propionate (TP) and exposed to ICW for 20 consecutive days. Sexual behavior was assessed on days 1, 5, 10, 15, and 20 and steroids were evaluated on day 20. Parameters of masculine sexual behavior were modified depending on the characteristics of each stressor. Mount, intromission and ejaculation latencies increased significantly, the number of mounts increased, and ejaculations decreased significantly in males exposed to EFS and to ICW but not in males exposed to IMB. Associated with these effects, testosterone decreased in the EFS and ICW groups on days 1, 15, and 20. However, corticosterone increased only in males exposed to ICW. In castrated males, TP treatment failed to block the effects of stress by ICW on sexual behavior and corticosterone. These results indicate that the effects of stress on sexual behavior depend on the characteristics of each stressor, and these effects, as well as the decrease in testosterone are not necessarily associated with the increase in corticosterone. The fact that testosterone treatment did not prevent the effects of stress on sexual behavior suggests that other mediators could be involved in the alterations of sexual behavior caused by stress.     

Changes in tissue water content measured with multiple-frequency bioimpedance and metabolism measured with 31P-MRS during progressive forearm exercise.

Multiple-frequency bioimpedance analysis (MFBIA) has been used to determine the cellular water composition in the human body. It is noninvasive and has demonstrated good correlations with other invasive measures of tissue water. However, the ability of this method to study transient changes in tissue water in specific muscle groups has not been explored. In this study, MFBIA was used to assess changes in forearm intracellular water (ICW), extracellular water (ECW), and total water (TW) in seven healthy volunteers during and after a progressive wrist flexion exercise protocol. In an identical trial, (31)P magnetic resonance spectroscopy ((31)P-MRS) was used to assess changes in intracellular pH and phosphocreatine (PCr). At the completion of exercise, forearm ICW increased 12.6% (SD 0.07, P = 0.003), TW increased 10.1% (SD 0.06, P = 0.005), and no significant changes were recorded for ECW. A significant correlation was found between the changes in intracellular pH and changes in ICW during exercise (r = -0.84, P = 0.018). With the use of regression analysis, average changes in P(i), PCr, and pH were found to predict changes in ICW (R(2) = 0.98, P = 0.005). In conclusion, MFBIA was sensitive enough to measure transient changes in the exercising forearm muscle. The changes seen were consistent with the hypothesis that intracellular acidification and PCr hydrolysis are important mediators of cellular osmolality and therefore may be responsible for the increased volume of water in the intracellular space that is often recorded after short-term high-intensity exercise.     

mRNA-targeted fluorescent in situ hybridization (FISH) of Gram-negative bacteria without template amplification or tyramide signal amplification

Technologies are needed to study gene expression at the level of individual cells within a population or microbial community. Fluorescent in situ hybridization (FISH) supplies high-resolution spatial information and has been widely applied to study microbial communities at the rRNA level. While mRNA-targeted FISH has been popular for studying gene expression in eukaryotic cells, very little success has been achieved with prokaryotes. At present, detection of specific mRNAs in individual prokaryotic cells requires the use of in situ RT-PCR or tyramide signal amplification (TSA). In this study we used DNA oligonucleotide probes labeled with a single near-infrared dye in FISH assays to detect multi-copy plasmid-based and endogenous mRNA molecules in Escherichia coli and Shewanella oneidensis MR-1. We took advantage of the fact that there is much less background signal produced by biological materials and support matrices in the near-infrared spectrum and thus long camera exposure times could be used. In addition, we demonstrate that a combination of probes targeting both rRNA and mRNA could be successfully employed within the same FISH assay. These results, as well as ongoing R&D improvements in NIR and infrared dyes, indicate that the FISH approach we demonstrated could be applied in certain environmental settings to monitor gene expression in mixed populations.     

Single- and multifrequency models for bioelectrical impedance analysis of body water compartments.

The 1994 National Institutes of Health Technology Conference on bioelectrical impedance analysis (BIA) did not support the use of BIA under conditions that alter the normal relationship between the extracellular (ECW) and intracellular water (ICW) compartments. To extend applications of BIA to these populations, we investigated the accuracy and precision of seven previously published BIA models for the measurement of change in body water compartmentalization among individuals infused with lactated Ringer solution or administered a diuretic agent. Results were compared with dilution by using deuterium oxide and bromide combined with short-term changes of body weight. BIA, with use of proximal, tetrapolar electrodes, was measured from 5 to 500 kHz, including 50 kHz. Single-frequency, 50-kHz models did not accurately predict change in total body water, but the 50-kHz parallel model did accurately measure changes in ICW. The only model that accurately predicted change in ECW, ICW, and total body water was the 0/infinity-kHz parallel (Cole-Cole) multifrequency model. Use of the Hanai correction for mixing was less accurate. We conclude that the multifrequency Cole-Cole model is superior under conditions in which body water compartmentalization is altered from the normal state.