Matthew R. Tubb

A three-dimensional homology model of lipid-free apolipoprotein A-IV using cross-linking and mass spectrometry.

Human apolipoprotein A-IV (apoA-IV) is a 46-kDa exchangeable plasma protein with many proposed functions. It is involved in chylomicron assembly and secretion, protection from atherosclerosis through a variety of mechanisms, and inhibition of food intake. There is little structural basis for these proposed functions due to the lack of a solved three-dimensional structure of the protein by x-ray crystallography or NMR. Based on previous studies, we hypothesized that lipid-free apoA-IV exists in a helical bundle, like other apolipoprotein family members and that regions near the N and C termini may interact. Utilizing a homobifunctional lysine cross-linking agent, we identified 21 intramolecular cross-links by mass spectrometry. These cross-links were used to constrain the building of a sequence threaded homology model using the I-TASSER server. Our results indicate that lipid-free apoA-IV does indeed exist as a complex helical bundle with the N and C termini in close proximity. This first structural model of lipid-free apoA-IV should prove useful for designing studies aimed at understanding how apoA-IV interacts with lipids and possibly with unknown protein partners.

Modulation of Apolipoprotein A-IV Lipid Binding by an Interaction between the N and C Termini

Apolipoprotein A-IV (apoA-IV) is a 376-amino acid exchangeable apolipoprotein made in the small intestine of humans. Although it has many proposed roles in vascular disease, satiety, and chylomicron metabolism, there is no known structural basis for these functions. The ability to associate with lipids may be a key step in apoA-IV functionality. We recently identified a single amino acid, Phe334, which seems to inhibit the lipid binding capability of apoA-IV. We also found that an intact N terminus was necessary for increased lipid binding of Phe334 mutants. Here, we identify Trp12 and Phe15 as the N-terminal amino acids required for the fast lipid binding seen with the F334A mutant. Furthermore, we found that individual disruption of putative amphipathic α-helices 3–11 had little effect on lipid binding, suggesting that the N terminus of apoA-IV may be the operational site for initial lipid binding. We also provide three independent pieces of experimental evidence supporting a direct intramolecular interaction between sequences near amino acids 12/15 and 334. This interaction could represent a unique “switch” mechanism by which apoA-IV changes lipid avidity in vivo.

Specific sequences in the N and C termini of apolipoprotein A-IV modulate its conformation and lipid association.

Apolipoprotein (apoA-IV) is a 376-residue exchangeable apolipoprotein that may play a number of important roles in lipid metabolism, including chylomicron assembly, reverse cholesterol transport, and appetite regulation. In vivo, apoA-IV exists in both lipid-poor and lipid-associated forms, and the balance between these states may determine its function. We examined the structural elements that modulate apoA-IV lipid binding by producing a series of deletion mutants and determining their ability to interact with phospholipid liposomes. We found that the deletion of residues 333–343 strongly increased the lipid association rate versus native apoA-IV. Additional mutagenesis revealed that two phenylalanine residues at positions 334 and 335 mediated this lipid binding inhibitory effect. We also observed that residues 11–20 in the N terminus were required for the enhanced lipid affinity induced by deletion of the C-terminal sequence. We propose a structural model in which these sequences can modulate the conformation and lipid affinity of apoA-IV.

Effect of intraperitoneal and intravenous administration of cholecystokinin-8 and apolipoprotein AIV on intestinal lymphatic CCK-8 and apo AIV concentration

CCK and apolipoprotein AIV (apo AIV) are gastrointestinal satiety signals whose synthesis and secretion by the gut are stimulated by fat absorption. Intraperitoneally administered CCK-8 is more potent in suppressing food intake than a similar dose administered intravenously, but the reason for this disparity is unclear. In contrast, both intravenous and intraperitoneally administered apo AIV are equally as potent in inhibiting food intake. When we compared the lymphatic concentration of CCK-8 and apo AIV, we found that neither intraperitoneally nor intravenously administered CCK-8 or apo AIV altered lymphatic flow rate. Interestingly, intraperitoneal administration of CCK-8 produced a significantly higher lymphatic concentration at 15 min than did intravenous administration. Intraperitoneal injection of apo AIV also yielded a higher lymphatic concentration at 30 min than did intravenous administration. Intraperitoneal administration of CCK-8 and apo AIV also resulted in a much longer period of elevated CCK-8 and apo AIV peptide concentration in lymph than intravenous administration. Furthermore, enzymatic activity of dipeptidyl peptidase IV (DPPIV) and aminopeptidase was higher in plasma than in lymph during fasting, and so, satiation peptides, such as CCK-8 and apo AIV in the lymph, are protected from degradation by the significantly lower DPPIV and aminopeptidase activity levels in lymph than in plasma. Therefore, the higher potency of intraperitoneally administered CCK-8 compared with intravenously administered CCK-8 in inhibiting food intake may be explained by both its higher concentration in lymph and the prolonged duration of its presence in the lamina propria.

Enhanced placental cholesterol efflux by fetal HDL in Smith–Lemli–Opitz syndrome

Previous studies from this laboratory have shown that maternal-derived cholesterol can be effluxed from trophoblasts to fetal HDL and plasma. We had the opportunity to study for the first time the ability of HDL and plasma from a fetus with the Smith-Lemli-Opitz syndrome (SLOS) to efflux cholesterol from trophoblasts. It was unclear whether cholesterol could be effluxed to fetuses with SLOS since lipoprotein levels are often very low. To answer this question, cord blood was collected from the placentas of an SLOS fetus and unaffected fetuses just after delivery. Plasma cholesterol concentrations were very low in the affected fetus; cholesterol, 7-dehydrocholesterol, and 8-dehydocholesterol concentrations were 14.1, 4.5, and 5.2 mg/dl, respectively. The HDL from the fetal SLOS effluxed approximately 50% more cholesterol from a trophoblast cell line, were smaller in size, and had a lower cholesterol to phospholipid ratio as compared to HDL from unaffected fetuses or adults. Plasma from the SLOS fetus effluxed cholesterol to a similar percentage as unaffected fetal plasma or adult plasma, possibly due to fewer HDL particles as demonstrated in previous SLOS patients. These novel data demonstrate that the cholesterol-deficient SLOS fetus is able to obtain cholesterol from trophoblasts at a time when cholesterol is playing a critical role in development, and has implications for design of treatments for cholesterol deficiency syndromes as well as understanding of prenatal cholesterol transport in humans.

The biotin-capture lipid affinity assay: a rapid method for determining lipid binding parameters for apolipoproteins

The lipid affinity of plasma apolipoproteins is an important modulator of lipoprotein metabolism. Mutagenesis techniques have been widely used to modulate apolipoprotein lipid affinity for studying biological function, but the approach requires rapid and reliable lipid affinity assays to compare the mutants. Here, we describe a novel method that measures apolipoprotein binding to a standardized preparation of small unilamellar vesicles (SUVs) containing trace biotinylated and fluorescent phospholipids. After a 30 min incubation at various apolipoprotein concentrations, vesicle-bound protein is rapidly separated from free protein on columns of immobilized streptavidin in a 96-well microplate format. Vesicle-bound protein and lipid are eluted and measured in a fluorescence microplate reader for calculation of a dissociation constant and the maximum number of potential binding sites on the SUVs. Using human apolipoprotein A-I (apoA-I), apoA-IV, and mutants of each, we show that the assay generates binding constants that are comparable to other methods and is reproducible across time and apolipoprotein preparations. The assay is easy to perform and can measure triplicate binding parameters for up to 10 separate apolipoproteins in 3.5 h, consuming only 120 μg of apolipoprotein in total. The benefits and potential drawbacks of the assay are discussed.

Diabetes in Homeless Persons: Barriers and Enablers to Health as Perceived by Patients, Medical, and Social Service Providers

The ways homelessness and diabetes affect each other is not well known. The authors sought to understand barriers and enablers to health for homeless people with diabetes as perceived by homeless persons and providers. The authors performed semistructured interviews with a sample of participants (seven homeless persons, six social service providers, and five medical providers) in an urban Midwest community. Data analysis was performed with the qualitative editing method. Participants described external factors (chaotic lifestyle, diet/food availability, access to care, and medications) and internal factors (competing demands, substance abuse, stress) that directly affect health. Social service providers were seen as peripheral to diabetes care, although all saw their primary functions as valuable. These factors and relationships are appropriately modeled in a complex adaptive chronic care model, where the framework is bottom up and stresses adaptability, self-organization, and empowerment. Adapting the care of homeless persons with diabetes to include involvement of patients and medical and social service providers must be emergent and responsive to changing needs.

Apolipoprotein A‐I structure in high‐density lipoproteins

High‐density lipoproteins (HDL) have attracted significant attention in recent years due to their apparent role in reducing the risk of coronary heart disease (CHD). There is convincing evidence that apolipoprotein (apo) A‐I, the main protein component in HDL, is a major mediator of HDL function that interacts with a host of plasma enzymes and cell surface proteins. ApoA‐I is highly adaptable and may associate with, and modulate the function of, different HDL subspecies. To better understand HDL function, studies have focused on apoA‐I structure in reconstituted HDL species that model different native subclasses of HDL. Powerful experimental strategies, such as X‐ray crystallography and mass spectrometry, combined with chemical cross‐linking and spectroscopy, have dramatically improved our understanding of apoA‐I structure in reconstituted HDL. In this review, we present an overview of the current understanding of apoA‐I structure and look forward to methodological developments in the near future that will allow the study of apoA‐I in native subspecies of HDL found in human plasma.

Case report of valproate-induced hypothermia in a patient with schizoaffective disorder.

To the Editor: Valproate is widely used to treat seizure disorders and prevent migraine headaches and is used as a mood stabilizer in patients with bipolar disorder. Valproate has an established adverse event profile. Two well-known valproate-induced adverse events are thrombocytopenia and hyperammonemia, which can lead to hematopoietic complications and delirium, respectively. However, hypothermia is another serious but significantly less documented adverse reaction to valproate treatment. While hypothermia is a serious medical emergency, a review of the limited published cases suggests that prompt discontinuation of valproate therapy results in restoration of normothermia. Return to thermal homeostasis typically occurs without ongoing medical complications arising from the period of drug-induced hypothermia. We report on such a case, in which prompt valproate discontinuation along with aggressive rewarming resulted in a full recovery for the patient. Case report. Ms A, a 48-year-old African American woman with a history of schizoaffective disorder (DSM-IV), hypertension, congestive heart failure, and type II diabetes mellitus, presented to the emergency department (ED) from the local state psychiatric facility in July 2008 having been found lethargic and hypothermic (axillary temperature 30°C [86°F]). The patient had a history of compulsive hand washing, water consumption, and showering. The psychiatric facility had noticed that, during the days prior to admission, the patient was abnormally docile. For at least 6 weeks prior to ED presentation, the patient had been on treatment with the following medications at stable doses: valproate (500 mg po qam, 1000 mg po qhs), risperidone (3 mg po bid, long-acting injection 50 mg IM every 2 wk), benztropine (0.5 mg po bid), haloperidol (5 mg/d po prn), furosemide (20 mg/d po), and magnesium (to treat furosemide-induced hypomagnesemia). The state psychiatric facility had suspected that the patient was “cheeking” her medicines, particularly the valproate, given lack of observed clinical efficacy. Approximately 3 weeks before the patient developed hypothermia, this suspicion was confirmed by the patients valproate level, which was barely detectable at < 1 ng/mL. More recently, the patient had been refusing valproate doses several times a week. Upon presentation to the ED, routine laboratory studies revealed low white blood cells at 1,700/μL and low platelets at 32,000/μL. The patients plasma valproate level was 28 μg/mL (normal range, 50–100 μg/mL) and plasma ammonia level was 50 μmol/L. In the ED, the rectal temperature was initially 30.5°C (86.9°F). At the time of presentation the patient was obtunded and her hypothermia was treated with a forced air warming device and warmed intravenous fluids. Following these interventions in the ED, her temperature improved to 33.3°C (91.9°F). At that time the patient was transferred to the medical intensive care unit (MICU) for further evaluation and monitoring. Upon admission to the MICU, all the patients antipsychotic medications were suspended due to concern for medication-induced adverse events. Given the patients current blood dyscrasias and altered mental status in the context of longstanding psychiatric illness, hematology/oncology and psychiatry consultations were obtained. Based on these consultations, the most likely diagnosis was drug-induced hypothermia and thrombocytopenia. Other investigatory studies that would have suggested disseminated intravascular coagulation or adrenal insufficiency were negative. Furthermore, the patients temperature continued to improve on day 2, to 36.8°C (98.2°F), following cessation of valproate therapy. The patient remained normothermic and became responsive by the third hospital day. The patients pancytopenia also improved following discontinuation of medications. Valproate was added to the patients allergy list after the psychiatry consult team reviewed the limited literature on valproate and hypothermia. For control of the patients primary psychiatric symptoms, risperidone 1 mg po bid was reinitiated, and she returned to her facility in stable condition. Upon return to her facility, the patient was placed on close staff monitoring to prevent her excessive water utilization. Routine surveillance of the patients vital signs showed that her temperature remained slightly low, but not hypothermic, at around 36°C (96.7°F). During this same period, laboratory studies demonstrated a platelet count within normal limits. Since the patients return to the state mental health facility, she has been maintained psychiatrically on treatment with risperidone, fluvoxamine, and lorazepam prn. A search of the archives of The Journal of Clinical Psychiatry (as well as The Primary Care Companion to The Journal of Clinical Psychiatry) revealed no articles involving valproate-induced hypothermia. Using the search terms valproate and hypothermia in the National Center for Biotechnology Informations PubMed returned only 17 articles. Only 5 of these articles, all case reports, describe human subjects and discuss the complication experienced by this patient. In 1984, Loscher and Vetter1 demonstrated a moderate hypothermic effect of valproate in rats in a study investigating the effects of drug-induced increases in γ-aminobutyric acid levels. The first case reports involving thermoregulation and valproate came in 2000 with 4 patients that developed hypothermia on valproate treatment and 1 patient who achieved heat tolerance while on valproate treatment.2 Three of these patients developed hypothermia shortly after being started on valproate therapy and their hypothermia resolved within days of the drugs discontinuation. The final patient discussed in the series had been taking valproate for 2 years and hypothermia did not develop until shortly after the patients coadministration of risperidone was discontinued. Nagarajan et al3 wrote a short response letter to that article adding a case of a child who developed hypothermia on valproate therapy. The child became hypothermic 2 days into valproate treatment and became normothermic within a week of discontinuation. In 2002, Longin et al4 reported 2 pediatric cases in which the patients tolerated valproate therapy but became hypothermic when topiramate was added to their medication regimen. Those cases occurred even though topiramate alone is not known to cause temperature regulation problems or exhibit a pharmacokinetic drug-drug interaction with valproate. Those patients also improved when valproate therapy was discontinued. Similarly, 2 cases were reported in which, although valproate levels were subtherapeutic, patients developed hypothermia when the dose of coadministered zotepine (an atypical antipsychotic) was increased.5 In those patients, gradual discontinuation of the zotepine was sufficient to regain normothermia. Finally, in 2005, the first case of severe hypothermia due to valproate overdose was reported.6 Contributing factors for the hypothermia in that case included very cold outside temperature and the patients immersion in cold water by nonmedical personnel due to concerns regarding potential alcohol intoxication. This patient fully recovered after aggressive rewarming. Since the patient under discussion was also on risperidone treatment, it is important to understand that drugs potential contribution to temperature regulation. In 2000, Oerther and Ahlenius7 demonstrated dose-dependent hypothermia in rats given risperidone. This effect was thought to be due to risperidones dopamine (D1) receptor agonism. A similar PubMed search revealed a single case report of risperidone-induced hypothermia.8 The authors related the hypothermia to risperidones preferential occupancy of serotonin-2 (5-HT2) receptors. Given all of the available data, risperidone, valproate, and compulsive water use all could have played a part in the development of the observed hypothermia in the patient under discussion. On the basis of the fact that her valproate dose was fluctuating leading up to the incident and her significant improvement after discontinuation of the valproate, we suspect that the valproate therapy was the most likely trigger for this patients hypothermia. This finding is similar to those from previously discussed case reports. The primary purpose of this report is to emphasize the emerging pattern of hypothermia induced by valproate. As this medication is often prescribed by primary care physicians, these physicians should, therefore, be familiar with this rare, but serious, adverse effect of valproate. This patient was fortunate to be in a long-term inpatient setting and monitored regularly. As such, she was transported promptly to the local ED and rewarming began as soon as possible. While complete recovery seems quite possible, early rewarming is important. The readership of the Companion may benefit from this reminder of the risk of hypothermia in their valproate-treated patients, particularly when valproate is used in combination with other psychotropic medications such as topiramate or risperidone.