Leveraging the Full Continuum of Care to Prevent Opioid Use Disorder.

Substance use disorder prevention programs are most effective when matched appropriately to the baseline risk of the population. Individuals who misuse opioids often have unique risk profiles different from those who use other substances such as alcohol or cannabis. However, most substance use prevention programs are geared toward universal audiences, neglecting key inflection points along the continuum of care. The HEAL Prevention Cooperative (HPC) is a unique cohort of research projects that represents a continuum of care, from community-level universal prevention to indicated prevention among older adolescents and young adults who are currently misusing opioids or other substances. This paper describes the theoretical basis for addressing opioid misuse and opioid use disorder across the prevention continuum, using examples from research projects in the HPC.

Osmometric analysis of quasi-indefinite mixed associations of the first kind.

Mixed associations of the type A + B----AB, A + AB----A2B, ..., A + Ai-1 B----AiB, ... are readily analyzed by osmometric methods. The equilibrium molar concentration of A, mA, is obtained very simply from mA = meq-m0B; here meq = c/Meqn is the equilibrium molar concentration of all associating species and m0B denotes the stoichiometric or original molar concentration of B. The quantity mB can then be obtained from methods developed by Steiner. The value of the binding polynomial lambda is given by lambda = m0B/mB; lambda is a function of mA only. In principle, one can evaluate the equilibrium constants (kA,B,etc.) by fitting lambda to the appropriate polynomial in mA of degree n (n = 2, 3, ...). The binding polynomial lambda is analogous to polynomials encountered in the analysis of self-associations. By making some simple assumptions one can develop four analogs of two sequential, equal equilibrium constant (SEK) or two attenuated equilibrium constant (AK) models. With the aid of r (the number average degree of binding), g (the osmotic coefficient), lambda, as well as mA and mB, one can evaluate the equilibrium constant or constants. The methods developed here can be extended to the nonideal case.

Molecular weights and molecular-weight distributions from ultracentrifugation of nonideal solutions.

Ultracentrifugation, membrane osmometry and capillary viscometry experiments have been performed on two dextran samples, which have molecular-weight distributions (MWDs) similar to those of dextrans used as blood plasma extenders. The manufacturer reported values of Mn and MW, determined by end group analysis and by light scattering, respectively. Our values of Mn, determined by osmometry, and MW, calculated from ultracentrifugal and viscometry experiments, agreed quite well with the manufacturer's results. Good agreement was obtained with values of MW and BLS (the light scattering second virial coefficient) obtained from sedimentation equilibrium experiments at different speeds using sector or nonsector-shaped centerpieces. Several ways of obtaining MW, MZ and BLS from sedimentation equilibrium experiments are presented. We have also shown how to obtain the speed-dependent term of the sedimentation equilibrium second virial coefficient. Both BLS and the speed-dependent nonideal terms could be used to correct the sedimentation equilibrium data, so that ideal values of d in c/d(r2) or dc/d(r2) could be estimated and used to obtain the MWDs of the dextran samples. Both Donnelly's and Scholte's methods were used with the sedimentation equilibrium data. With both methods, unimodal MWDs were encountered, which gave good agreement with the manufacturer's MWDs, obtained by a combination of analytical gel chromatography and light scattering. Uncorrected sedimentation equilibrium data gave MWDs quite different from the manufacturer's results. The MWD calculated from the differential distribution of sedimentation coefficients also gave a unimodal MWD, but this MWD did not give a good agreement with the sedimentation equilibrium results or with the manufacturer's results.

Self-association of sodium cholate in isotonic saline solutions.

The self-association of dialyzed solutions of sodium cholate in isotonic saline solutions has been studied by vapor pressure osmometry and sedimentation equilibrium. These studies were carried out at 25, 31 and 37 degrees C. In all experiments the self-association could be described as a two-equilibrium constant, indefinite self-association in which odd species beyond monomer were absent. The plots of M1/Mna or M1/Mwa vs. c were quite smooth with no sharp breaks; this suggested that there were no critical phenomena. The temperature dependence of the self-association was quite small. Our results are in accord with other studies on sodium cholate which indicate that the self-association involves several species, and that it is not a monomer-n-mer self-association.

Osmotic pressure measurements of ovalbumin and lysozyme mixtures.

Ovalbumin and lysozyme have been reported to undergo a mixed association in solutions of low ionic strength. Osmotic pressure experiments were performed on ovalbumin and on lysozyme solutions in 0.06 M cacodylate buffer (I = 0.02, pH = 5.8) at 30 and at 37 degrees C. The individual proteins did not undergo any self-associations at either temperature; these measurements indicated that each of the solutions was nonideal. Osmotic pressure experiments on three blends of lysozyme and ovalbumin at 30 and 37 degrees C could be interpreted in two ways. One interpretation was that a nonideal, nonassociating mixture of A and B was present; for the three solutions the mixed nonideal term BAB was negative. A negative nonideal term is usually interpreted as indicating an association. The other interpretation of the data was as a quasi-ideal mixed association of the type A + B in equilibrium AB.

Self-association of beta-lactoglobulin c in acetate buffers.

The self-association of beta-lactoglobulin C at pH 4.65 (23 degrees C) in acetate buffer has been studied at various temperatures, 10, 16, 20 and 25 degrees C, by a series of sedimentation equilibrium experiments. Two different buffers were used. Buffer I with an ionic strength of 0.1 consisted of 0.1M acetic acid and 0.1M sodium acetate; buffer II had 0.1M KCl in addiation so that its ionic strength was 0.2. The variation of the apparent weight average molecular weight, Mwa, with the total solute concentration, c, was characteristic of a self-association. In contrast to the behavior of beta-lactoglobulin A in acetate buffer, the association of beta-lactoglobulin C did not proceed beyond dimer. Furthermore, within the experimental error, the self-association of beta-lactoglobulin C was independent of temperature and ionic strength; all experimental data could be put on the same Mwa (or M1/Mwa) vs. c plot! Several models were used to test the self-association, and a monomer--dimer association with K2 = 2.10 X 10(3) dl/g and BM1 =- 1.2 X 10(-2) dl/g seemed to give a good description of the M1/Mwa vs. c curve.

Analysis of self-associations from partition isotherms.

Self-associations can be studied from the measurements of the partition of the self-associating solute between two immiscible liquids. The apparent partition coefficient, Kapp, is proportional to the ratio of the apparent weight fraction of monomer, fa, in each phase. If one assumes that the Adams-Fujita convention for the activity coefficients of the self-associating species applies, then fa is related to Mna and Mwa, the apparent values of the number and weight average molecular weights, respectively; and one can use previously developed methods to analyze the self-association. In order to use the method, one must make an independent study at the same temperature of one of the phases by an appropriate thermodynamic method, such as vapor pressure osmometry or sedimentation equilibrium. Then one can test the other phase for the type of self-association present and evaluate the equilibrium constant or constants (ki) and the nonideal term (BM1) from the partition data. One can also evaluate the partition coefficient (Kpar). From these measurements, one can obtain the free energy (delta G0) for the association in each phase and for the transfer between phases. Temperature-dependence studies will provide the enthalpy (delta H0) or entropy (delta S0) of self-association or transfer. This method should be quite useful for studying small molecules of biological importance.

Mixed association of cholesterol with methyl cholate and methyl lithocholate in chloroform solutions.

The concentration-dependent mixed association behavior of cholesterol with methyl cholate (MeC) and methyl lithocholate (MeLC) in chloroform at 37 degrees C has been studied by vapor pressure osmometry (VPO). This study is part of a larger project to investigate the effect of number and position of hydroxyl-bearing steroids. Using theories developed by Adams and by Steiner, the model and appropriate parameters for the nonideal mixed associations were elucidated. For the MeLC/cholesterol system, no mixed association was observed. For the MeC/cholesterol system, both methods of analysis indicate that a nonideal AB complex formation occurs. The best parameters to explain the experimental data are kAB = 0.04 1/g; BAB (the nonideal term) = 1.5 X 10(-5) 1 mol g-2.

Analysis of various indefinite self-associations of the AK type.

Although we and others have developed equations to analyze for some indefinite self-associations that might be encountered, it is felt in some cases that these models, known as the sequential, equal equilibrium constant (SEK) models, might overestimate the size of aggregates encountered at higher solute concentrations. Thus, Garland and Christian proposed two attenuated equilibrium constant (AK) models that might overcome this problem. Their methods were restricted to ideal solutions and to osmometric procedures. We have removed these restrictions, and we have developed equations for analyzing four AK models that might be encountered. Various tests to aid in distinguishing these models are presented. These procedures have been tested with two simulated examples of a Type III AK indefinite self-association.

Analysis of various indefinite self-associations.

Two methods have been developed for the analysis of four types of indefinite self-associations. Unlike previous treatments by others, the procedures can be applied to nonideal cases. The two methods were first tested with simulated data, and it was found that one could indeed distinguish between the four types of indefinite self-associations. For a more realistic test, sedimentation equilibrium experiments were performed on solutions of beta-lactoglobulin A at 16 degrees C in 0.15 ionic strength acetate buffer, pH 4.65. The self-association of the beta-lactoglobulin A was best described by either method as a sequential indefinite self-association having two equilibrium constants and one second virial coefficient.

The temperature-dependent self-association of adenosine 5'triphosphate in 0.154 M NaCl.

The disodium salt of adenosine 5'triphosphate (ATP), dissolved in and dialyzed against isotonic saline, undergoes a temperature-dependent self-association. Self-association increases with decreasing temperature. The technical achievement that made these experiments possible was the development and availability of a hollow fiber dialyzer with a low molecular weight (200) cutoff. Several models were tested to describe the experimental data, and a monomer-dirner-trimer association seemed to describe the data best- Both the dimerization and trimerization were exothermic; values of the thermodynamic functions for these reactions are reported. The self-association reaction for ATP is much stronger than that observed for purine and cyridine, as well as for various nucleosides whose self-association in aqueous solutions has been studied.

Sedimentation coefficients of self-associating species. II. Tests with a simulated example and with beta-lactoglobulin A.

If sedimentation equilibrium and sedimentation velocity experiments are performed on a self-associating solute under the same solution conditions, it is possible to evaluate the sedimentation coefficients (si) of the self-associating species and the usual concentration dependence parameter (g or gs). We have tested some of these methods with simulated examples. A more critical test is to use real data. Sedimentation equilibrium experiments with beta-lactoglobulin A at 20 degrees C in 0.2 M glycine buffer (pH 2.46) indicated that a nonideal monomer-dimer association was present. Sedimentation velocity experiments were performed on beta-lactoglobulin A under the same conditions. Using data from both sets of experiments we were able to evaluate s1, s2, g and gs using two different models for swa, the apparent weight average sedimentation coefficient. The empirical model for swa developed by Weirich et al. [1] gave better variance than did the model for swa developed by Gilbert and his co-workers [2-5]. Using a simulated monomer-dimer association mimicking a system having higher sedimentation coefficients than beta-lactoglobulin A did, we were able to show that one could not obtain s2 from tangents to the plot of 1/swa vs. c in the high concentration region. The methods developed here for sedimentation coefficients can be applied to other experiments in which a weight average property (or its apparent value) of a self-associating solute is measured, provided the appropriate thermodynamic experiments are done under the same solution conditions.

Application of osmometric methods to the study of lipids, lipoproteins, and apolipoproteins.

Membrane and vapor pressure osmometry are two colligative methods that can be useful in lipid research. The former method can be used to study proteins or other macromolecules whose molecular weight lies between 20,000 to 1,000,000. Vapor pressure osmometry is useful with smaller molecules having a molecular weight of 10,000 or less. These techniques can be used in aqueous or nonaqueous solutions. They are rapid, precise, nondestructive, and require relatively small amounts of material. These techniques provide information about the state of aggregation and also about interactions of lipids, lipoproteins, and apolipoproteins in solution. We will show how membrane osmometry can be used to study solutions of lipoproteins and apolipoproteins. The application of vapor pressure osmometry to the study of biologically important lipids such as cholesterol, cholesteryl esters, and bile salts is shown.

Iron-induced myocardial and hepatic lysosomal abnormalities in the guinea pig.

The guinea pig model of iron overload, described in the preceding article, was used to investigate the mechanism of excess iron toxicity in hepatic and cardiac tissues. Effects of iron overload on both lysosomal membrane fragility and membrane peroxidation were studied. The free activity of selected myocardial and hepatic lysosomal enzymes, in addition to serum activity, was measured in guinea pigs treated with iron dextran (0.25, 0.5, 1.0, and 2.0 g Fe/kg body weight); controls received dextran. Levels of malondialdehyde were also determined in whole homogenates of heart and liver in animals loaded with 0.5 and 1.5 g Fe/kg of iron dextran. Results indicated that the free activity of hepatic glucosaminidase (p < 0.05) and beta-glucuronidase (p < 0.05) were significantly elevated at all levels of iron loading; hepatic acid phosphatase was increased at all but the lowest iron dose. Similarly, increased serum glucosaminidase activity was observed (p < 0.01) at all dose levels. When compared to pooled controls, the free activity of myocardial glucosaminidase was also elevated (p < 0.05) at all levels of loading. However, myocardial acid phosphatase was increased only at the highest iron dose (p < 0.01). Increased malondialdehyde was measured at the high iron dose (1.5 g Fe/kg) in whole homogenates of both heart and liver (p < 0.01). We conclude that iron loading in this model profoundly alters the stability of hepatic and myocardial lysosomal membranes; furthermore, changes in serum glucosaminidase activity may be reflective of modified tissue lysosomal properties. Elevated levels of malondialdehyde in whole homogenates suggest that iron-mediated lipid peroxidation may be responsible in part for enhanced lysosomal membrane fragility.

Morphologic investigations of the guinea pig model of iron overload.

We have developed a guinea pig model of iron overload toxicity. Animals were administered intraperitoneal iron dextran 3 times a week to achieve total body iron load of 0.25, 0.5, 1.0, 1.5, and 2.0 g Fe/kg body weight in less than 30 days. Quantitation of tissue iron levels with atomic absorption indicated increased iron deposition in liver and heart of the iron-loaded guinea pigs (p < 0.001). Additionally, the iron-loaded pigs demonstrated decreased nuclear magnetic resonance spectroscopy T1 relaxation times in both liver and heart (p < 0.001). Serum iron, total body iron capacity, and transferrin saturation values were also determined in guinea pigs treated with 0.25, 0.5, and 1.0 g Fe/kg body weight. Serum iron and total iron-binding capacity were significantly increased at 0.5 and 1.0 g Fe/kg; transferrin saturation was elevated at 0.25 and 1.0 g Fe/kg. kg. Histologic examination of liver, heart, and bone marrow as well as ultrastructural studies on liver and heart confirmed increased iron deposition in treated animals. At the low iron dose level of 0.5 g Fe/kg, liver iron particles were primarily confined to Kupffer cells with minimal hepatocellular localization. Increased hepatocellular iron deposition was observed with larger doses of loaded iron. Myocardial iron was most prominent in interstitial cells of the epicardium, endocardium, myocardium, and coronary adipose tissue. Ultrastructurally, the presence of iron particles in perinuclear, membrane-bound structures (consistent with lysosomes) was confirmed using x-ray microanalysis. These morphological studies suggest that in this animal model siderosis of hepatic mononuclear phagocyte and myocardial interstitial cells may be the initial lesions leading to further biochemical and functional abnormalities. Correlation between tissue iron measurements and both light and electron microscopic changes, presented in this report, serve to introduce the iron-loaded guinea pig as a model for the study of iron-induced tissue damage.