Two domains of the smoothelin-like 1 protein bind apo- and calcium–calmodulin independently

The smoothelin-like 1 protein (SMTNL1) is a modulator of smooth and skeletal muscle contractility and can bind to calmodulin and tropomyosin. Calmodulin is the major calcium sensor of eukaryotic cells and it can cycle between calcium-free (apo-CaM) and calcium-bound (Ca-CaM) forms. Bioinformatic screening of the SMTNL1 sequence predicted a second CaM-binding region (CBD1) that is located N-terminal to the previously defined apo-CaM-binding site (CBD2). Pull-down assays, surface plasmon resonance, isothermal calorimetry and NMR techniques were used to determine that CBD1 associated preferentially to Ca-CaM while CBD2 bound preferentially to apo-CaM. Mutation of hydrophobic residues abolished Ca-CaM-binding to CBD1 while acidic residues in CBD2 were necessary for apo-CaM-binding to CBD2. The dissociation constant (K_d) for Ca-CaM-binding to a CBD1 peptide was 26 ∗ 10^− 6M while the value for binding to a longer protein construct was 0.5 ∗ 10^− 6 M. The binding of SMTNL1 to both apo-CaM and Ca-CaM suggests that endogenous CaM is continuously associated with SMTNL1 to allow for quick response to changes in intracellular calcium levels. We also found that the intrinsically disordered N-terminus of SMTNL1 can reduce binding to apo-CaM and increase binding to Ca-CaM. This finding suggests that an additional CaM-binding region may exist and/or that intramolecular interactions between the N-terminus and the folded C-terminus reduce apo-CaM-binding to CBD2. Intriguingly, CBD1 is located close to the SMTNL1 phosphorylation site and tropomyosin-binding region. We discuss the possibility that all three signals are integrated at the region surrounding CBD1.     

Chronic beryllium disease: an updated model interaction between innate and acquired immunity

During the last decade, there have been concerted efforts to reduce beryllium (Be) exposure in the workplace and thereby reduce potential cases of this occupational lung disorder. Despite these efforts, it is estimated that there are at least one million Be-exposed individuals in the U.S. who are potentially at risk for developing chronic beryllium disease (CBD). Previously, we reviewed the current CBD literature and proposed that CBD represents a model interaction between innate and acquired immunity (Sawyer et al., Int Immunopharmacol 2:249–261, 2002). We closed this review with a section on “future directions” that identified key gaps in our understanding of the pathogenesis of CBD. In the intervening period, progress has been made to fill in some of these gaps, and the current review will provide an update on that progress. Based on recent findings, we provide a new hypothesis to explain how Be drives sustained chronic inflammation and granuloma formation in CBD leading to progressive compromised lung function in CBD patients. This paradigm has direct implications for our understanding of the development of an immune response to Be, but is also likely applicable to other immune-mediated lung diseases of known and unknown etiology.     

ERK phosphorylation and tumor necrosis factor-alpha production by monocytes are persistent in response to immobilized IgG

Engagement of Fcγ receptors on leukocytes by immune complexes induces both cytokine production and immune complex internalization. The relationship between these processes is unclear. In many disease states, Fcγ receptors encounter their ligands in deposited forms that cannot be readily internalized. In this study, we examined the kinetics of ERK1/2 phosphorylation and TNF-α secretion in primary human monocytes in response to soluble heat-aggregated IgG or surface-bound IgG, to mimic soluble immune complexes and tissue-deposited IgG, respectively. Soluble aggregated IgG induced transient signaling, leading to peak phosphorylation of ERK1/2 by 15 min and peak TNF-α levels by 1 h, whereas surface-bound IgG caused sustained responses over the course of several hours. Treatment with the vacuolar ATPase inhibitor bafilomycin led to increased persistence of ERK1/2 phosphorylation in response to aggregated IgG. When monocytes were incubated with both soluble aggregated IgG and surface-bound IgG simultaneously, ERK1/2 phosphorylation was transient. These results suggest that Fcγ receptor internalization is an important step in termination of inflammatory signaling, and that small immune complexes can exert an overall down-modulatory effect when encountered in the presence of immobilized IgG.     

Beryllium binding at neutral pH: the importance of the Be-O-Be motif

Beryllium speciation at physiological conditions is critical to understanding chronic beryllium disease (CBD). The MHC-class II receptor alleles that have been linked to CBD have more than six carboxylates in a short 20 amino acid segment of the binding pocket and it has been suggested that beryllium may bind within the MHC-class II receptor via the carboxylates. Previous reports also show that citric acid binds beryllium significantly stronger than similar carboxylate ligands such as tartaric acid and is one of the few ligands that can compete with hydrolysis to solubilize beryllium across the entire pH range at molar concentrations. We have characterized the binding of Be to citric acid and shown using a combination of NMR, mass spectrometry and ligand competition studies that Be2L and Be4L2 species dominate. A Be-O-Be linkage with the bridging oxygen coming from the aliphatic alcohol is critical to the stability of the complex. We show through competition experiments that the most stable Be-O-Be arrangement has one Be in a five-member ring and the other Be in a six-member ring. The unusual deprotonation of an aliphatic alcohol (pK(a) = 18) at neutral pH has significant ramifications on the potential interactions of Be with biological ligands such as carbohydrates and Ser and Thr residues.     

Exploring the role of short-course cyclosporin a therapy in preventing homograft valve calcification after transplantation

This study was designed to explore the role of short-course cyclosporin A therapy in preventing calcification. Homograft valves heterotopically allografted onto abdominal aorta from SD to Wistar rats. The expression of CD25, CD40L, CD71, calcium content and morphological change were observed. In control group, expression of immune indices got maximal at early stage postoperatively, and then gradually declined, remained at low level 12 weeks afterwards. In test group with Cyclosporin A, the expression of immune indices were lower than that of control group at 2–4 weeks postoperatively, but no significant difference was found 8 weeks afterwards. The calcification began from 4 weeks postoperatively, increased gradually and reached highest level at 12 weeks. In test group calcium content was much lower from 4 to 16 weeks postoperatively. It is concluded that cyclosporine A treatment can prevent calcification of homograft valves because it inhibited immune response at early stage after transplantation.     

Beryllium Increases the CD14dimCD16+ Subset in the Lung of Chronic Beryllium Disease

CD14^dimCD16+ and CD14^brightCD16+ cells, which compose a minor population of monocytes in human peripheral blood mononuclear cells (PBMC), have been implicated in several inflammatory diseases. The aim of this study was to investigate whether this phenotype was present as a subset of lung infiltrative alveolar macrophages (AMs) in the granulomatous lung disease, chronic beryllium disease (CBD). The monocytes subsets was determined from PBMC cells and bronchoalveolar lavage (BAL) cells from CBD, beryllium sensitized Non-smoker (BeS-NS) and healthy subjects (HS) using flow cytometry. The impact of smoking on the AMs cell phenotype was determined by using BAL cells from BeS smokers (BeS-S). In comparison with the other monocyte subpopulations, CD14^dimCD16+ cells were at decreased frequency in PBMCs of both BeS-NS and CBD and showed higher HLA-DR expression, compared to HS. The AMs from CBD and BeS-NS demonstrated a CD14^dimCD16+phenotype, while CD14^brightCD16+ cells were found at increased frequency in AMs of BeS, compared to HS. Fresh AMs from BeS-NS and CBD demonstrated significantly greater CD16, CD40, CD86 and HLA-DR than HS and BeS-S. The expression of CD16 on AMs from both CBD and BeS-NS was downregulated significantly after 10μM BeSO₄ stimulation. The phagocytic activity of AMs decreased after 10μM BeSO₄ treatment in both BeS-NS and CBD, although was altered or reduced in HS and BeS-S. These results suggest that Be increases the CD14^dimCD16+ subsets in the lung of CBD subjects. We speculate that Be-stimulates the compartmentalization of a more mature CD16+ macrophage phenotype and that in turn these macrophages are a source of Th1 cytokines and chemokines that perpetuate the Be immune response in CBD. The protective effect of cigarette smoking in BeS-S may be due to the low expression of co-stimulatory markers on AMs from smokers as well as the decreased phagocytic function.     

Inmunosenescencia prematura en ratones triple-transgénicos para la enfermedad del Alzheimer

Introduction

A deterioration of the neuroimmunoendocrine network has been observed in Alzheimer's disease (AD). However, the peripheral immune response has hardly been investigated in this pathology. Since some immune function parameters have been established as good markers of the rate of ageing, and can predict longevity, the aim of the present work was to study some of these functions in splenic leucocytes in transgenic mice for AD of different ages.

Material and methods

Young female (4 ± 1 months), adult (9 ± 1 months), and mature (12 ± 1 months) triple-transgenic mice for AD (3 xTgAD) and non-transgenic (NTg) control mice of the same ages were used. The chemotaxis, the anti-tumour activity of «natural killer» (NK) cells and the lymphoproliferative response in the presence of the mitogens concanavalin A and lipopolysaccharide, functions that decrease with age, were determined in splenic leucocytes. In addition, the differences in lifespan between 3 xTgAD and NTg were studied in parallel using other animals, until their death through natural causes.

Results

In 3 xTgAD, with respect to NTg, chemotaxis decreased at all ages studied, whereas in lymphoproliferative response this reduction was shown at 4 months and 9 months. NK activity was diminished only in young 3 xTgAD with respect to NTg. The 3 xTgAD showed a shorter lifespan than the NTg control group.

Conclusions

The 3 xTgAD mice show a premature immunosenescence, which could explain their early mortality. The determination of these immune functions at peripheral level could serve as a marker of the progression of the Alzheimer's disease.     

Chronic Beryllium Disease: Revealing the Role of Beryllium Ion and Small Peptides Binding to HLA-DP2

Chronic Beryllium (Be) Disease (CBD) is a granulomatous disorder that predominantly affects the lung. The CBD is caused by Be exposure of individuals carrying the HLA-DP2 protein of the major histocompatibility complex class II (MHCII). While the involvement of Be in the development of CBD is obvious and the binding site and the sequence of Be and peptide binding were recently experimentally revealed [1], the interplay between induced conformational changes and the changes of the peptide binding affinity in presence of Be were not investigated. Here we carry out in silico modeling and predict the Be binding to be within the acidic pocket (Glu26, Glu68 and Glu69) present on the HLA-DP2 protein in accordance with the experimental work [1]. In addition, the modeling indicates that the Be ion binds to the HLA-DP2 before the corresponding peptide is able to bind to it. Further analysis of the MD generated trajectories reveals that in the presence of the Be ion in the binding pocket of HLA-DP2, all the different types of peptides induce very similar conformational changes, but their binding affinities are quite different. Since these conformational changes are distinctly different from the changes caused by peptides normally found in the cell in the absence of Be, it can be speculated that CBD can be caused by any peptide in presence of Be ion. However, the affinities of peptides for Be loaded HLA-DP2 were found to depend of their amino acid composition and the peptides carrying acidic group at positions 4 and 7 are among the strongest binders. Thus, it is proposed that CBD is caused by the exposure of Be of an individual carrying the HLA-DP2*0201 allele and that the binding of Be to HLA-DP2 protein alters the conformational and ionization properties of HLA-DP2 such that the binding of a peptide triggers a wrong signaling cascade.     

Gender-specific cold responses induce a similar body-cooling rate but different neuroendocrine and immune responses

This study investigated whether there are any gender differences in body-heating strategies during cold stress and whether the immune and neuroendocrine responses to physiological stress differ between men and women. Thirty-two participants (18 men and 14 women) were exposed to acute cold stress by immersion to the manubrium level in 14 °C water. The cold stress continued until rectal temperature (T_RE) reached 35.5 °C or for a maximum of 170 min. The responses to cold stress of various indicators of body temperature, insulation, metabolism, shivering, stress, and endocrine and immune function were compared between men and women. During cold stress, T_RE and muscle and mean skin temperatures decreased in all subjects (P < 0.001). These variables and the T_RE cooling rate did not differ between men and women. The insulative response was greater in women (P < 0.05), whereas metabolic heat production and shivering were greater (P < 0.05) in men. Indicators of cold strain did not differ between men and women, but men exhibited larger changes in the indicators of neuroendocrine (epinephrine level) and in immune (tumor necrosis factor-α level) responses (both P < 0.05). The results of the present study indicated that men exhibited a greater metabolic response and shivering thermogenesis during acute cold stress, whereas women exhibited a greater insulative response. Despite the similar experience of cold strain in men and women, the neuroendocrine and immune responses were larger in men. Contrary to our expectations, the cooling rate was similar in men and women.     

The kinetics of local cytokine and galectin expression after challenge infection with the gastrointestinal nematode, Haemonchus contortus

Gastrointestinal nematode parasites undergo several developmental stages within their mammalian host, each presenting different antigenic challenges to the immune system. To examine the expression of different immune mediators over time, biopsy samples were collected from the cannulated abomasum (true stomach) of immune sheep at several times after a challenge infection with Haemonchus contortus L3s. IL-5 and IL-13 mRNA expression levels were significantly increased above saline-challenged control levels at 5 and 7 days post challenge, while IL-4 showed an earlier peak at day 2 post challenge. IL-5, IL-13 and IL-4, as well as IFN-γ mRNA levels, peaked at 7 days before decreasing to non-significant levels at 28 days post challenge. TNF-α followed a similar profile while there was a slight increase in TGF-β in both control and challenged sheep. There was a significant increase in galectin-14 mRNA in the L3 challenged compared with the saline challenged group at 7 days while both galectin-11 protein and mRNA levels increased significantly by day 3 post challenge, peaking at 5-7 days post challenge. Distinct correlations were observed between these immune parameters at different times after L3 challenge. The galectin-14 protein level at day 2 post challenge was the only measured mediator significantly negatively correlated with worm burden. These studies highlight the dynamic nature of the immune response during parasite infection and the need to consider the different life cycle stages involved.     

Neopterin, a marker of immune response, and 8-hydroxy-2'-deoxyguanosine, a marker of oxidative stress, correlate at high age as determined by automated simultaneous high-performance liquid chromatography analysis of human urine

Using an established high-performance liquid chromatography (HPLC) method based on anion exchange chromatography, fraction collection, and electrochemical detection, the oxidative DNA damage marker 8-hydroxy-2′-deoxyguanosine (8-OH-dG) can be analyzed rapidly and precisely in human urine samples. In addition, by ultraviolet (UV) detection, it was shown recently that it is possible to simultaneously analyze creatinine and 7-methylguanine (m⁷Gua), an RNA degradation product, in urine. By adding a fluorescence detector to the HPLC system, we now report that it is also possible to detect pteridins such as neopterin and biopterin. The fluorescence detection was evaluated in detail for neopterin, an immune response and tumor marker. The urinary content of neopterin, assessed by using the HPLC method, was verified with a commercial neopterin enzyme-linked immunosorbent assay (ELISA) kit as indicated by the high correlation between the two methods (r = 0.98). In urinary samples from 58 young healthy individuals (male and female nonsmokers, ages 19-39 years), it was found that there was no significant correlation (r = −0.04) between the levels of 8-OH-dG and neopterin (as normalized to urinary creatinine levels). In contrast, in urinary samples from 60 old healthy individuals (male and female nonsmokers, ages 60-86 years), there was a significant correlation (r = 0.47) found between the levels of 8-OH-dG and neopterin (as normalized to urinary creatinine levels). These findings strongly indicate that the higher level of immune response that was correlating with old age contributes significantly to the higher level of oxidative damage as assessed in the form of 8-OH-dG. Using this type of HPLC system, it is possible to evaluate oxidative DNA damage and immune response simultaneously using the respective urinary markers. These data may contribute to understanding of the pathophysiology of diseases such as infections and tumor progression where both oxidative stress and immune response occur simultaneously.     

Elastase B of Pseudomonas aeruginosa stimulates the humoral immune response in the greater wax moth, Galleria mellonella

The role of Pseudomonas aeruginosa elastase B in activation of the humoral immune response in Galleria mellonella larvae was investigated. The results of our study showed that elastase B injected at a sublethal concentration was responsible for eliciting the humoral immune response in G. mellonella larvae. The insects exhibited increased antibacterial activity, namely, we observed appearance of antimicrobial peptides and a higher level of lysozyme in cell-free hemolymph. Elastase B seems to be a more potent elicitor than thermolysin because similar maximal antibacterial activity levels were observed at a 5-fold lower concentration. We also demonstrated that there were differences in the kinetics of induction of antimicrobial activity between thermolysin and elastase B. The maximum level was observed 18 h post challenge of thermolysin and 38 h after injection of elastase B. It was also shown that, 24 h after elastase injection, the relative levels of apoLp-III in the hemolymph significantly increased in comparison with control G. mellonella larvae. The activation of immune responses in metalloproteinase-challenged larvae involved synthesis of metalloproteinase inhibitors which increased the survival rates of insects both against the lethal dose of thermolysin as well as against viable pathogenic bacterial cells of P. aeruginosa.     

Hollow fiber-based liquid phase microextraction with factorial design optimization and gas chromatography–tandem mass spectrometry for determination of cannabinoids in human hair

A new method, based on hollow fiber liquid-phase microextraction (HF-LPME) and gas chromatography–tandem mass spectrometry (GC–MSMS), was developed for determination of Δ⁹-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in samples of human hair. Since hair is a solid matrix, the samples were subjected to alkaline digestion using NaOH. The aqueous solutions obtained were extracted using a 6 cm polypropylene fiber (600 μm i.d., 200 μm wall thickness, 0.2 μm pore size) for each extraction. A 2⁵⁻¹ fractional factorial design for screening, and a central composite design for optimization of significant variables, was applied during development of the extraction method. The variables evaluated were the type of extraction solvent, pH, stirring speed, extraction time, and acceptor phase volume. The optimized conditions for the proposed extraction procedure were 10 mg of hair sample; 20 μL of butyl acetate; aqueous (pH 14) donor phase containing 6.8% NaCl; 600 rpm stirring speed; 20 min extraction time. A linear response was obtained in the ranges 1–500 pg mg⁻¹ (CBD and CBN) and 20–500 pg mg⁻¹ (THC), with regression coefficients >0.99. Precision, determined as the relative standard deviation, was 3.3–8.9% (intra-day) and 4.4–13.7% (inter-day). Absolute recoveries varied in the ranges 4.4–4.8% (CBD), 7.6–8.9% (THC) and 7.7–8.2% (CBN). Limits of detection (LOD, S/N = 3) and quantification (LOQ, S/N = 10) were 0.5–15 pg mg⁻¹ and 1–20 pg mg⁻¹, respectively. The method was successfully used to determine CBD, THC and CBN in hair samples from patients in a drug dependency rehabilitation center. Concentrations varied in the ranges 1–18 pg mg⁻¹ (CBD), 20–232 pg mg⁻¹ (THC) and 9–107 pg mg⁻¹ (CBN), confirming the suitability of the method for monitoring studies.     

Improved cold starch hydrolysis with urea addition and heat treatment at subgelatinization temperature

We explore how the presence of urea can influence the kinetics of amylolysis, with a long-term objective of developing practical and energy efficient bioconversion protocols. In this study, triticale and corn starches were hydrolyzed by a granular starch hydrolyzing enzyme with or without addition of urea and a pre-heating treatment at subgelatinization temperature. Differential scanning calorimetry showed that the gelatinization parameters of triticale and corn starches were negatively correlated with the urea concentration in the starch suspension. Addition of urea did not significantly affect starch amylolysis by the granular starch hydrolyzing enzyme at 30 °C. However when pre-heating at a higher yet sub-gelatinization temperature (50 °C for triticale and 61 °C for corn, 5 °C below the onset of starch gelatinization) for 30 min, the presence of urea greatly facilitated the amylolysis of both tricticale and corn starches. Scanning electron microcopy revealed starch granule mophological changes to a porous structure in residual starch granules/fragments rich in resistant starch. This means that the amylolysis pattern in the presence of urea was fundamentally changed, and urea disrupts starch hydrogen bonds effectively with heating treatment at a sub-gelatinization temperature. This treatment combination increased both starch hydrolysis rate and extent. Since extra energy was not necessary to gelatinize starch, this method may benefit starch and bio-enthanol industries to reduce the costs of starch hydrolysis.     

Acculturation, childhood trauma and the cortisol awakening response in Mexican-American adults

Exposure to chronic and traumatic stress has been associated with the dysregulation of crucial stress response systems. Acculturation has been associated with unique forms of chronic psychosocial stress. The purpose of this study was to examine the effects of exposure to early traumatic stress and acculturation on dysregulation of the cortisol awakening response (CAR) in Mexican-American adults. Salivary cortisol samples were collected at awakening and 30, 45, and 60 min thereafter, on two consecutive weekdays from 59 healthy Mexican-American adult males (26) and females (33), ages 18-38 years. Participants were assessed for level of acculturation and exposure to early trauma. Data were analyzed using a mixed effects regression model with repeated measures at four time points. Mixed effects regression results indicated a significant Early Trauma × Time interaction (p = .0029) and a significant Acculturation × Time interaction (p = .0015), after controlling for age and sex. Subsequent analyses of the interaction of Trauma × Acculturation × Time showed that more than minimal exposure to either risk factor was associated with attenuation of the awakening cortisol response (p = .0002). Higher levels of acculturation with greater Anglo-orientation were associated with attenuation of the CAR in Mexican-American adults. Both moderate and higher levels of exposure to early trauma were associated with an attenuated CAR. However, greater exposure to both risk factors was only incrementally worse than exposure to either one.     

The effect of dietary nickel on the immune responses of Spodoptera litura Fabricius larvae

By exposing Spodoptera litura Fabricius larvae to nickel (Ni) in artificial diets for successive three generations, we investigated the impacts of the dietary Ni on growth and immune response of the fifth and sixth instar larvae at 24 h intervals. The time of newly moulted fifth instar larvae was labelled as 0 h. After exposure to 5 mg/kg Ni for two generations, Ni exposure significantly improved larval phenoloxidase activity and encapsulation grade in fifth instar larvae when compared to controls, except for encapsulation grade at 72-120 h in the second generation. However, higher concentrations of Ni (≥10 mg/kg) only significantly reduced encapsulation grade at 72-120 h. In the third generation, insects given higher dietary levels of Ni (≥10 mg/kg) showed lower immune responses and retarded relative growth rate (RGR) compared to controls, but those exposed to lower Ni levels (≤5 mg/kg) had a significantly improved encapsulation grade at 24-72 h. Larvae at lower Ni level (≤5 mg/kg) treatments had significantly higher RGR in comparison with that in controls. There was no significant difference in food relative consumption rate (RCR) and RGR among any treatment of the fifth instar larvae in three successive generations. These results indicated that the type and extent of effects on growth and immune responses of S. litura varied with the Ni concentrations and exposure periods.     

Molluskan fasciclin-1 domain-containing protein: Molecular characterizationand gene expression analysis of fasciclin 1-like protein from disk abalone (Haliotis discus discus)

Cell-to-cell contacts play a key role in multicellular systems and organisms. Fasciclin-1 (FAS-1) is a lipid-linked membrane associated glycoprotein that is a member of a newly recognized family of cell adhesion molecules sharing features with the immunoglobulins, cadherins, integrins, and selectins. Here, we report the identification and molecular characterization of a novel FAS-1 domain-containing cDNA from disk abalone (Haliotis discus discus), including its gene expression profile and immune response to bacterial stimuli and tissue injuries. Designated as Abfac1, the 909 bp open reading frame (ORF) encodes 303 amino acid (aa) residues with a predicted molecular mass of 33 kDa and isoelectric (pI) value of 4.9. The aa sequence contains two FAS-1 domains and three conserved regions, FRa motif, H-box, and FRb motif. Phylogenetic analysis showed the closest relation to Jellyfish cell adhesion protein. In healthy abalone, Abfac1 expression is highest in hepatopancreas followed by mantle and lowest in digestive gland. In immune-stimulated abalones, relative Abfac1 mRNA expression was increased in hemocytes by ~ 11-fold at 48 h after the Vibrio parahaemolyticus infection, by 3.1-fold at 6 h after the Listeria monocytogenes infection and by ~ 9-fold at 6 h after the LPS injection. Similarly, tissue injuries caused significant increase of relative mRNA expression by 3.5-fold in hemocytes and by ~ 10-fold in mantle at 12 h post-injury. These results suggest that the novel member of the FAS-1 domain-containing protein family, Abfac1, may be involved in immune response and cell adhesion in disk abalone.     

A liquid chromatography method for quantifying caffeine dissolution from pharmaceutical formulations into colloidal,fat-rich media

A simple and rapid high-performance liquid-chromatography method is presented that permits quantification of caffeine in colloidal fat emulsions proposed as new ‘biorelevant’ dissolution media (Intralipid™ and various milks). Using a mobile phase of 0.1 M sodium acetate (pH 4.0) and acetonitrile (89.5:10.5, v/v) at 1 ml min⁻¹, the drug and internal standard (7-β-hydroxyethyltheophylline) were eluted within 8 min. Caffeine extraction was undertaken by protein precipitation in ice-cold 12% (w/v) trichloroacetic acid and centrifugation at 10,000 rpm for 15 min. This simple extraction method generated caffeine recovery values (corrected for % fat content) of 75.4 ± 1.4–100.6 ± 5.5%. The limit of detection was within the range 0.25–0.4 μg ml⁻¹ and linearity was demonstrated in each medium up to 125 μg ml⁻¹. Precision was <11.5% RSD and intra- and inter-day accuracy was 93.4–109.3%. The validated method was applied to in vitro USP dissolution tests in milk which compared the kinetics of caffeine release from (i) extended release matrices containing hydroxypropyl methylcellulose (HPMC) and (ii) an immediate release commercial analgesic tablet. Good reproducibility was obtained in both extended and immediate release dissolution tests. The method provides high-throughput quantification of this common drug in fat emulsions used as biorelevant dissolution media.