Preschool to School in Autism: Neuropsychiatric Problems 8 Years After Diagnosis at 3 Years of Age.

The study presents neuropsychiatric profiles of children aged 11 with autism spectrum disorder, assessed before 4.5 years, and after interventions. The original group comprised a community sample of 208 children with ASD. Parents of 128 participated-34 with average intellectual function, 36 with borderline intellectual function and 58 with intellectual disability. They were interviewed using the Autism-Tics, AD/HD and other Comorbidities interview. Criteria for a clinical/subclinical proxy of ASD were met by 71, 89 and 95 %, respectively. Criteria for at least one of ASD, AD/HD, Learning disorder or Developmental Coordination Disorder were met by 82, 94 and 97 %. More than 90 % of children with a preschool diagnosis of ASD have remaining neuropsychiatric problems at 11, despite early intervention.

Prenatal ultrasound and childhood autism: long-term follow-up after a randomized controlled trial of first- vs second-trimester ultrasound.

OBJECTIVE: To analyze whether the frequency of autism spectrum disorder (ASD) in a cohort of Swedish children differs between those exposed to ultrasound in the 12(th) week and those exposed to ultrasound in the 18(th) week of gestation. METHODS: The study cohort consisted of approximately 30 000 children born between 1999 and 2003 to mothers who had been randomized to a prenatal ultrasound examination at either 12 or 18 weeks' gestation as part of the framework for a study on nuchal translucency screening. The outcome measure in the present study was the rate of ASD diagnoses among the children. Information on ASD diagnoses was based on data from the Swedish social insurance agency concerning childcare allowance granted for ASD. RESULTS: Between 1999 and 2003, a total of 14 726 children were born to women who underwent a 12-week ultrasound examination and 14 596 to women who underwent an 18-week ultrasound examination. Of these, 181 (1.2%) and 176 (1.2%) children, respectively, had been diagnosed with ASD. There was no difference in ASD frequency between the early and late ultrasound groups. CONCLUSIONS: Women subjected to at least one prenatal ultrasound examination at either 12 or 18 weeks' gestation had children with similar rates of ASD. However, this result reflects routine care 10-15 years ago in Sweden. Today, higher intensity ultrasound scans are performed more frequently, at earlier stages during pregnancy and for non-medical purposes, implying longer exposure time for the fetus. This change in the use of ultrasound necessitates further follow-up study of the possible effects that high exposure to ultrasound during the gestational period has on the developing brain. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.

Academic performance of adolescents with ADHD and other behavioural and learning problems -a population-based longitudinal study.

AIM: To study academic performance (final grades at the age of 16 years) in individuals with i) attention-deficit/hyperactivity disorder (ADHD) and ii) other learning and/or behavioural problems. METHODS: Of a total population of 591 children, originally assessed at the age of 10-11 years, it was possible to obtain final grades for 536 16-year-olds (in grade 9). Those fulfilling the criteria for ADHD/sub-threshold ADHD (n = 39) and those with 'Behaviour and Learning Problems' (BLP group), (n = 80) and a comparison group (n = 417) were contrasted. RESULTS: The ADHD and BLP groups had a significantly lower total mean grade at the age of 16 years than the comparison group. In addition, the ADHD and BLP groups also qualified for further studies in the upper secondary school to a significantly lesser extent than the controls (72%, 68% and 92%, respectively). All IQ measures (at the age of 10-11 years) were positively correlated with the overall grade after grade 9, with especially strong correlations for verbal capacity. CONCLUSION: ADHD and similar problems entail a risk of underachievement at school. The results indicate that pupils with ADHD underachieve in the school situation in relation to their optimal cognitive capacity. The contextual situation and the particular requirements should be considered in order for adequate educational measures to be undertaken.

The 22q11 deletion syndrome candidate gene Tbx1 determines thyroid size and positioning.

Thyroid dysgenesis is the major cause of congenital hypothyroidism in humans. The underlying molecular mechanism is in most cases unknown, but the frequent co-incidence of cardiac anomalies suggests that the thyroid morphogenetic process may depend on proper cardiovascular development. The T-box transcription factor TBX1, which is the most probable gene for the 22q11 deletion syndrome (22q11DS/DiGeorge syndrome/velo-cardio-facial syndrome), has emerged as a central player in the coordinated formation of organs and tissues derived from the pharyngeal apparatus and the adjacent secondary heart field from which the cardiac outflow tract derives. Here, we show that Tbx1 impacts greatly on the developing thyroid gland, although it cannot be detected in the thyroid primordium at any embryonic stage. Specifically, in Tbx1-/- mice, the downward translocation of Titf1/Nkx2.1-expressing thyroid progenitor cells is much delayed. In late mutant embryos, the thyroid fails to form symmetric lobes but persists as a single mass approximately one-fourth of the normal size. The hypoplastic gland mostly attains a unilateral position resembling thyroid hemiagenesis. The data further suggest that failure of the thyroid primordium to re-establish contact with the aortic sac is a key abnormality preventing normal growth of the midline anlage along the third pharyngeal arch arteries. In normal development, this interaction may be facilitated by Tbx1-expressing mesenchyme filling the gap between the pharyngeal endoderm and the detached thyroid primordium. The findings indicate that Tbx1 regulates intermediate steps of thyroid development by a non-cell-autonomous mechanism. Thyroid dysgenesis related to Tbx1 inactivation may explain an overrepresentation of hypothyroidism occurring in patients with the 22q11DS.

Culturing and characterization of astrocytes isolated from juvenile rainbow trout (Oncorhynchus mykiss).

An access to brain cell cultures from fish would enable screening of possible neurotoxic chemicals contaminating the aquatic environment. In the present study, a protocol for a successful routine isolation and culturing of brain cells from juvenile rainbow trout was worked out. The coating material was shown to be of importance for cell proliferation. Cells grow better on a surface coated with laminin than on those coated with poly-L-lysine (PLL), poly-D-lysin (PDL) or poly-L-ornithine (PLO). The best cell growth was obtained on double-coated surfaces (PLL, PDL or PLO plus laminin). On such a culture substrate and with a seeding density of 1 x 10(7) cells/cm(2) confluence was obtained within 3-4 weeks at an incubation temperature of 18 degrees C. Approximately 95% of the cells were identified as astrocytes on the basis of a positive staining with antibodies against the astrocyte specific glial protein (GFAP). No oligodendrocytes or fibroblasts were identified in the cultures, and despite several efforts, neurons did not grow under the culture conditions used. When challenged with ligands known to awake a calcium transient in mammalian astrocytes, 44% of the cells responded to ATP with an increase in [Ca 2+](i), 38% to norepinephrine, 27% to 5-hydroxytryptamine, 7% to histamine and 6% to glutamate. Kainate, quisqualate and gamma-aminobutyric acid did not awake a calcium transient in the cells. Using a proper protocol, it is thus quite easy to get an almost pure culture of astrocyte, whereas neurones proved to very difficult to culture.

Expression of caspase-1 in synovial membrane-like interface tissue around loosened hip prostheses.

Caspase-1 expression in synovial membrane-like interface tissue (SMLIT) around loosened hip prostheses and osteoarthritic synovial samples was studied. Caspase-1 mRNA was found in SMLIT and synovial tissue. There is no difference in the copy numbers of caspase-1 mRNA between these samples. Both precursor and active forms of caspase-1 proteins appeared in these samples, but the number of positive cells was higher in SMLIT than in synovial tissue. Double labeling revealed that most caspase-1-positive cells were macrophages and fibroblasts. In the lining-like layers and deep stroma of SMLIT, many cells were double positive for active caspase-1 and interleukin-1 beta (IL-1beta). In contrast, the number of active caspase-1/IL-18 double-positive cells was very low. We conclude that caspase-1 synthesis is increased in SMLIT. Caspase-1 can be involved in implant loosening by processing IL-1beta precursor into its mature form, which is a potent osteoclast-activating factor and a major proinflammatory mediator.

Glucose metabolism and pulsatile insulin release from isolated islets.

The effects of metabolic inhibition on insulin release and the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) were studied in individually perifused pancreatic islets from ob/ob mice. The modest basal secretion in the presence of 3 mmol/l glucose was pulsatile with a frequency of approximately 0.2/min, although [Ca(2+)](i) was stable at approximately 100 nmol/l. Introduction of 11 mmol/l glucose resulted in large amplitude oscillations of [Ca(2+)](i) and almost 20-fold stimulation of average secretion manifested as increased amplitude of the insulin pulses without change in frequency. Inhibition of glycolysis with iodoacetamide or mitochondrial metabolism with dinitrophenol or antimycin A reduced glucose-stimulated secretion back to basal levels with maintained pulsatility. The [Ca(2+)](i) responses to the metabolic inhibitors were more complex, but in general there was an initial peak and eventually sustained elevation without oscillations. When introduced in the presence of 3 mmol/l glucose, the metabolic inhibitors tended to increase the amplitude of the insulin pulses, although the simultaneous elevation in [Ca(2+)](i) occurred without oscillations. The data indicate that pulsatile secretion is regulated by factors other than [Ca(2+)](i) under basal conditions and after metabolic inhibition. Although pulsatile secretion can be driven by oscillations in metabolism when [Ca(2+)](i) is stable, it was not possible from the present data to determine whether insulin pulses have a glycolytic or mitochondrial origin.

Comparative responsiveness of measures of pain and function after total hip replacement.

OBJECTIVE: To compare the responsiveness of the Functional Assessment System (FAS), the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and the Medical Outcomes Study 36-item Short Form (SF-36) in patients with osteoarthritis (OA) scheduled for total hip replacement. METHOD: Twenty patients with a mean age at surgery of 72.6 years, with primary OA of the hip, were investigated preoperatively and at 3, 6, and 12 months postoperatively with the FAS, WOMAC, and SF-36. The responsiveness was calculated as standardized response mean, effect size, and relative efficiency. RESULTS: The pain and function scores of WOMAC and SF-36 showed greater responsiveness than FAS at 3 months. These differences remained at 6 and 12 months postoperatively. The differences between these 3 outcome measures were found to be similar using several methods for calculating responsiveness. CONCLUSION: Self-administered questionnaires like WOMAC and SF-36 are more responsive measures of pain and function than range of motion, performance tests, and observer-administered questions (FAS) following total hip replacement.

Glucose-regulated pulsatile insulin release from mouse islets via the K(ATP) channel-independent pathway.

OBJECTIVE: Regulation of insulin release by glucose involves dual pathways, including or not inhibition of ATP-sensitive K(+) channels (K(ATP) channels). Whereas the K(ATP) channel-dependent pathway produces pulsatile release of insulin it is not clear whether the independent pathway also generates such kinetics. DESIGN AND METHODS: To clarify this matter, insulin secretion and cytoplasmic Ca(2+) ([Ca(2+)](i)) were studied in perifused pancreatic islets from ob/ob mice. Insulin release was measured by ELISA technique and [Ca(2+)](i) by dual-wavelength fluorometry. RESULTS: Insulin secretion was pulsatile (0.2--0.3/min) at 3 mmol/l glucose when [Ca(2+)](i) was low and stable. Stimulation with 11 mmol/l of the sugar increased the amplitude of the insulin pulses with maintained frequency and induced oscillations in [Ca(2+)](i). Permanent opening of the K(ATP) channels with diazoxide inhibited glucose-stimulated insulin secretion back to basal levels with maintained pulsatility despite stable and basal [Ca(2+)](i) levels. Increase of the K(+) concentration to 30.9 mmol/l in the continued presence of diazoxide and 11 mmol/l glucose restored the secretory rate with maintained pulsatility and caused stable elevation in [Ca(2+)](i). Simultaneous introduction of diazoxide and elevation of K(+) augmented average insulin release almost 30-fold in 3 mmol/l glucose with maintained pulse frequency. Subsequent elevation of the glucose concentration to 11 and 20 mmol/l increased the release levels. After prolonged exposure to diazoxide, elevated K(+) and 20 mmol/l glucose, the pulse frequency decreased significantly. CONCLUSIONS: Not only glucose signaling via the K(ATP) channel-dependent but also that via the independent pathway generates amplitude-modulated pulsatile release of insulin from isolated islets.

Amplitude modulation of pulsatile insulin secretion by intrapancreatic ganglion neurons.

Neuron activity and insulin release were measured simultaneously from 33 preparations of intrapancreatic canine ganglia and pancreatic parenchyma adjacent to the ganglia. The electrical activity of single neurons of the ganglia was recorded with intracellular microelectrodes, and insulin release from the attached islets was determined with an enzyme-linked immunosorbent assay. Insulin release was 62 +/- 18 fmol preparation/min in the presence of 10 mmol/l glucose and pulsatile (3.7 +/- 0.4 min/pulse). Corresponding measurements of neuronal electrical activity showed a stable membrane potential of -53.5 +/- 0.6 mV. Short, high-frequency (20 Hz) preganglionic nerve stimulation evoked action potentials and, in 46% of the preparations, a threefold rise in the insulin secretory rate associated with increased amplitude of the insulin pulses. The effects were blocked by 10 micromol/l tetrodotoxin (TTX). In other preparations, continuous low-frequency (0.05-0.5 Hz) preganglionic nerve stimulation evoked action potentials and, in 50% of the preparations, a gradual increase of insulin release associated with augmentation of insulin pulse amplitude without alteration of the duration. The effects were blocked by 50 micromol/l hexamethonium (HEX). In the remaining preparations, no change in insulin release was observed during nerve stimulation. In the absence of stimulation, neither TTX nor HEX affected the membrane potential or insulin secretion. These first simultaneous measurements of intrapancreatic ganglion activity and insulin secretion are consistent with amplitude modulation of pulsatile insulin secretion induced by changes in electrical activity in a population of intrapancreatic ganglion neurons.

Preattentive bias for emotional information in panic disorder with agoraphobia.

A combined emotional Stroop and implicit memory (tachistoscopic identification) task with 3 types of words (panic-related, interpersonal threat, and neutral words) and 2 exposure conditions (subliminal, supraliminal) was administered to 35 patients with panic disorder and 35 age- and sex-matched controls. The patients showed Stroop interference for panic-related words both sub- and supraliminally and a similar but not equally robust effect on interpersonal threat words. On the tachistoscopic identification task, the patients identified more panic-related words than the controls did but showed no implicit memory bias effect. The patients' subliminal Stroop interference for panic-related words was found to correlate with trait anxiety and depression, although not with anxiety sensitivity.

Pulsatile insulin release: role of cytoplasmic Ca2+ oscillations.

Oscillations of plasma insulin are essential for the hypoglycaemic effect of the hormone. Disturbance and partial loss of these oscillations occur during the development of Type 2 diabetes, in association with down-regulation of insulin receptors and insulin resistance. Oscillations with a frequency similar to that of plasma insulin have been observed in the cytoplasmic Ca2+ concentration ([Ca2+]i) of pancreatic beta cells, indicating that the ion plays a role in generating insulin pulses. Studies of individual islets have revealed that oscillations of [Ca2+]i and insulin release are synchronous. However, insulin release is also pulsatile under conditions in which [Ca2+]i is stable. These results support the notion that variations in the ATP/ADP ratio are sufficient to induce pulsatile insulin release. Under physiological conditions, this pulsatility may depend on the synergistic effects of ATP/ADP and [Ca2+]i oscillations.

Pulsatile insulin release from pancreatic islets with nonoscillatory elevation of cytoplasmic Ca2+.

The relationship between insulin release and cytoplasmic Ca2+ concentration ([Ca2+]i) was studied in isolated pancreatic islets from ob/ob mice. Although [Ca2+]i was low and stable in the presence of 3 mM glucose, basal insulin release exhibited low amplitude pulsatility, with a frequency of 0.32 +/- 0.04 min-1. Depolarization by raising K+ from 5.9 to 30.9 mM or by the addition of 1 mM tolbutamide caused a pronounced initial insulin pulse followed by declining pulses, but there was no change in frequency. This decline in amplitude of the insulin pulses was prevented in similar experiments performed in the presence of 11 mM glucose. Corresponding measurements of [Ca2+]i in islets exposed to tolbutamide or the high K+ concentration revealed stable elevations without oscillations. Although the [Ca2+]i level is an important determinant for the rate of secretion, the results indicate that pulsatile insulin release does not always depend on [Ca2+]i oscillations. It is suggested that cyclic generation of ATP may fuel pulsatile release under conditions when [Ca2+]i remains stable.

Oscillatory signaling and insulin release in human pancreatic beta-cells exposed to strontium.

Oscillatory signaling and insulin release were studied in isolated pancreatic islets and beta-cells obtained from human cadaveric organ donors. Taking advantage of Sr2+ as an analog for Ca2+, it was possible to demonstrate glucose-induced rhythmic activity in individual beta-cells identified by immunostaining. Glucose-induced slow oscillations of Sr2+ (frequency, 0.1-1.0/min) were sometimes seen at a sugar concentration as low as 3 mM. Addition of 20 nM glucagon resulted in a broadening of the oscillations or in their transformation into sustained elevation. Moreover, the presence of glucagon resulted in the appearance of short transients of Sr2+, which disappeared after exposure to the intracellular Ca2+-adenosine triphosphatase inhibitor thapsigargin. Digital image analyses indicated that slow oscillations can be synchronized among cells in small aggregates and intact islets. The rhythmic activity in the glucose-stimulated beta-cell had its counterpart in pulsatile insulin release when single islets were perifused with a Sr2+-containing medium. It is concluded that the human beta-cell has oscillatory signaling for insulin release similar to that observed in experimental animals.

Functional bioactive recombinant acylation stimulating protein is distinct from C3a anaphylatoxin.

Acylation stimulating protein (ASP) acts upon adipose tissue to stimulate triglyceride synthesis and glucose transport. The aim of the present study was to produce recombinant ASP and to measure its bioactivity. The cDNA region of the parent complement C3 sequence coding for ASP (C3adesArg) was cloned and expressed in E. coli. Bioactivity of the purified recombinant material was tested by determining its effect on triglyceride synthesis, glucose transport, and competition binding assays. In standard assays, concentrations of 5.5 microM recombinant ASP (rASP) stimulated triglyceride synthesis comparably to plasma ASP (pASP): 228% versus 237%, respectively, in 3T3 preadipocytes and 568% versus 440% in human differentiated adipocytes. rASP also increased glucose transport in L6 myocytes (163% at 10 microm rASP) and in human differentiated adipocytes (334% rASP vs. 329% pASP at 5 microM). rASP competitively displaced radiolabeled plasma ASP from high affinity association with the cell surface in both human differentiated adipocytes and 3T3 preadipocyte fibroblasts. Furthermore, immunoprecipitation of rASP and pASP with a specific monoclonal antibody abolished stimulation of cellular triglyceride synthesis. Lastly, we contrasted the structure:function activities of the arginated (C3a) and desarginated (ASP) proteins. The lipogenic activity and the anaphylatoxic activity result from distinct structural domains of the polypeptides. Thus rASP retains full biologic ASP activity and may provide a tool to study structure-function relationships in this physiologic system.

Pulsatile insulin release from mouse islets occurs in the absence of stimulated entry of Ca2+.

Pancreatic islets are known to respond to a raise of the glucose concentration with Ca2+ -induced 2-3-min pulses of insulin release. The reports of cyclic variations of circulating insulin in the fasting state made it important to explore whether insulin release is also pulsatile in the absence of stimulated entry of Ca2+. Individual pancreatic islets were isolated from a local colony of ob/ob mice and perifused under conditions allowing dual wavelength recordings of the cytoplasmic Ca2+ concentration ([Ca2+]i) with fura-2 and measurements of insulin with ELISA technique. At 3 mM of glucose, [Ca2+]i remained at a stable low level, but insulin was released in pulses with a frequency of 0.41+/-0.02 min-1, determined by Fourier transformation of original and autocorrelated data. Pulses of basal insulin release were also seen when glucose was omitted and 1 microM clonidine or 400 microM diazoxide was added to a glucose-free medium. The results indicate that pulsatile insulin release can be generated in the absence of stimulated entry of Ca2+. A tentative explanation for this phenomenon is inherent fluctuations in the ATP production of the beta cells.

Elution of lipoprotein fractions containing apolipoproteins E and A-I in size exclusion on Superose 6 columns is sensitive to mobile phase pH and ionic strength.

Separation of lipoproteins secreted from McA-RH7777 (rat hepatoma) cells by Superose 6 column size-exclusion chromatography, using PBS buffer (NaCl 150 mM, sodium phosphate 10 mM, pH 7.5, EDTA 1 mM), produced apolipoprotein (apo) E or A-I profiles that did not correlate with lipoproteins separated by density ultracentrifugation. By density ultracentrifugation, apoE and apoA-I were mostly (> 90%) confined to high-density lipoproteins (HDL, d = 1.063-1.023 g/ml), but by chromatography apoE and apoA-I were recovered in all lipoprotein classes, including low-density lipoproteins (LDL), HDL, and post-HDL. Moreover, the elution volume of phenol red on Superose 6 greatly exceeded the total column volume. These discrepancies were attributable to pH and ionic strength effects. In low ionic strength, high pH buffer (Tris 25 mM, pH 8.3), elution volumes of lipoproteins, albumin, and phenol red were minimized. Elution volumes increased 25-70% when buffer pH was lowered at constant ionic strength (Tris 25 mM, pH 7.4) or when ionic strength was increased at constant pH (Tris 25 mM, pH 8.3, NaCl 500 mM). Altered phase partition appeared to cause the altered elution volumes, since recovery (measured as analyte peak area), resolution (measured as peak width at half height), and column void volume varied little from buffer to buffer. In Superose 6 size-exclusion chromatography with PBS buffer, then, elution volumes vary with pH and ionic strength. We propose that TBE buffer (Tris-borate 89 mM, pH 8.3, EDTA 2 mM) may produce fewer artefacts than PBS. With TBE there were (i) better correlation between size-exclusion and ultracentrifugal fractions, (ii) lower elution volumes, and (iii) less ¿smearing¿ of McA-RH7777 apoE and apoA-I containing lipoprotein bands.

Carboxyl-terminal truncation impairs lipid recruitment by apolipoprotein B100 but does not affect secretion of the truncated apolipoprotein B-containing lipoproteins.

Human apolipoprotein (apo) B plays an obligatory role in the assembly and secretion of hepatic triglyceride-rich lipoproteins. Investigation of the truncated human apoB variants associated with hypobetalipoproteinemia has suggested that both size and secretion of apoB-containing lipoproteins may be reduced by carboxyl-terminal truncation. To examine the role of the carboxyl terminus of apoB in the assembly and secretion of hepatic lipoproteins, we have generated rat hepatoma McA-RH7777 cells that synthesize and secrete the full-length human apoB100 and the truncated forms B94, B88, B80, B72, and B60. In the resulting lipoproteins, particle density was inversely related to the logarithm of apoB length, ranging from 1.019 g/ml for apoB100 to 1.06 g/ml for B60. Furthermore, particle diameter (as determined by non-denaturing gel electrophoresis) was directly correlated with apoB length, ranging from 21.4 nm for apoB100 to 17.7 nm for B60. The relationship between apoB length and particle geometry was best defined by a linear correlation between length and core volume; a 10% decrease in apoB length resulted in an approximately 13% decrease in core volume. These observations, which are in agreement with the observations of aberrant lipoproteins in hypobetalipoproteinemia, suggest that lipid recruitment by apoB is progressively reduced by carboxyl-terminal truncation. However, pulse-chase studies indicated that carboxyl-terminal truncation did not impair apoB secretion. The recombinant human apoB forms were secreted as efficiently as endogenous rat apoB100; approximately 20% of total newly synthesized apoB72, B80, or B100 was secreted at the end of the chase. Intracellular degradation of newly synthesized apoB was observed for both the truncated human and the endogenous rat proteins. These data suggest that the low apoB levels in hypobetalipoproteinemia might not be caused by impaired secretion of the truncated apoB proteins.

Discrete carboxyl-terminal segments of apolipoprotein E mediate lipoprotein association and protein oligomerization.

The carboxyl terminus of apolipoprotein (apo) E is required for lipoprotein association and for tetramer formation. To correlate these roles with specific regions within the carboxyl terminus, a series of apoE3 variants with carboxyl-terminal truncations at residues 266, 244, 223, and 191 were expressed in Escherichia coli. As determined by gel permeation and sedimentation equilibrium centrifugation, the four truncated variants were monomeric in solution. Compared to native apoE3 (299 residues), all had reduced affinity for lipoproteins, as assessed by incubation of 125I-labeled proteins with plasma followed by fractionation of lipoprotein classes by gel filtration. The 266-residue variant associated with very low density lipoproteins and high density lipoproteins, but was partly non-lipoprotein-bound (25% of total). Shorter variants, with 244 or fewer residues, did not associate with very low density lipoproteins and only associated slightly (approximately 20%) with high density lipoproteins, with the major portion non-lipoprotein-bound (65-73%). After these proteins were injected into rabbits, the clearance rate was proportional to the plasma level of non-lipoprotein-bound protein. These results indicate lipoprotein association modulates the clearance of apoE, residues within the segment 267-299 are critical for apoE tetramerization and facilitate lipoprotein association, and residues within the segment 245-266 also contribute to lipoprotein association.