PubMed Health⌕ Search

Biomedical subjects

Y Israel

Publications and source records attributed to Y Israel.

At least 37 records · Page 2Linked to original sources
PubMed1997

Serum IgA, IgG, and IgM antibodies directed against acetaldehyde-derived epitopes: relationship to liver disease severity and alcohol consumption.

Chronic ethanol ingestion has been suggested to trigger the formation of antibodies that recognize acetaldehyde-protein condensates. In this study, assays for immunoglobulin (Ig) A, IgG, and IgM antibodies to acetaldehyde-derived adducts were performed on sera of 140 alcohol consumers, 19 patients with nonalcoholic liver disease (NALD), 35 healthy nondrinking controls, and 10 nondrinking patients with IgA or IgG myeloma. Anti-acetaldehyde (Ach)-adduct antibodies of each Ig isotype were found from the alcohol abusers. In alcoholic liver disease (ALD, n = 86) IgA titers were elevated in 69% of the patients. These titers were significantly higher than those from patients with NALD (P < .001), nondrinking controls (P < .001), or heavy drinkers (n = 54) without any clinical and biochemical signs of liver disease (P < .001). In contrast, anti-adduct IgG titers were significantly elevated both in ALD and in heavy drinkers as compared with patients with NALD (P < .001) or nondrinking controls (P < .01 and P < .05, respectively). The anti-adduct immunoglobulin (Ig)A, IgG, and IgM titers in patients with alcoholic liver disease (ALD) correlated with the combined clinical and laboratory index of liver disease severity (r(s) = .497, P < .001; r(s) = .361, P < .01; and r(s) = .322, P < .01). Anti-adduct IgA titers also correlated with serum bilirubin (r = .768, P < .001) and interleukin 6 (r = .504, P < .001). Anti-adduct IgG titers were, in turn, found to correlate with the presence of inflammation (P < .01) and necrosis (P < .01). During follow-up studies of individual patients, parallel changes were observed in the anti-adduct IgG titers, disease severity, and serum markers of fibrogenesis. The present results provide evidence that antibodies representing distinct Ig classes directed against acetaldehyde (Ach)-derived adducts of proteins are formed in alcoholic patients showing an association with the severity of liver disease. The follow-up data also support an association between such immune responses and the aggravation of liver disease.

Acetaldehyde↗
PubMed1996

Cytochrome P4502E1 hydroxyethyl radical adducts as the major antigen in autoantibody formation among alcoholics.

BACKGROUND & AIMS: We have previously reported that alcoholics have increased titers of immunoglobulins reacting with protein adducts of hydroxyethyl free radicals. Because hydroxyethyl radicals are produced during ethanol metabolism by liver microsomes, the aim of this study was to determine whether such antibodies recognize microsomal proteins complexed with hydroxyethyl radicals. METHODS: Liver microsomal proteins reacting with the anti-hydroxyethyl radical antibodies were characterized by an enzyme-linked immunosorbent assay and Western blotting. RESULTS: Alcoholic cirrhotics, but not patients with nonalcoholic cirrhosis or healthy subjects, had increased serum levels of immunoglobulin G and A directed against antigens produced in microsomes incubated with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and ethanol. Such immunoreactivity was completely blocked when microsomes were incubated with ethanol in the presence of the spin-trapping agent 4-pyridyl-1-oxide-t-butyl nitrone or by preincubating the sera with hydroxyethyl radical-bound human albumin. Immunoblotting of proteins from human liver microsomes incubated with NADPH and ethanol showed that 86% of the sera from alcoholic cirrhotics reacted with a 52-kilodalton protein, whereas variable reactivity was observed with proteins of 78, 60, and 40 kilodaltons, respectively, The 52-kilodalton protein was identified by immunoblotting and immunoprecipitation as ethanol-inducible cytochrome P4502E1. CONCLUSIONS: Antibodies from alcoholic cirrhotics specifically recognized hydroxyethyl radical-cytochrome P4502E1 adducts, suggesting the possible implication of these antigens in the development of autoimmune reactions in alcoholic liver disease.

Adult↗
PubMed1996

Sensitivity and specificity of carbohydrate-deficient transferrin as a marker of alcohol abuse are significantly influenced by alterations in serum transferrin: comparison of two methods.

Despite a number of investigations suggesting the value of carbohydrate-deficient transferrin (CDT) as a marker of alcohol abuse, a variety of issues on the applicability of CDT measurements in clinical settings have remained unexplored. Earlier studies in this field have focused on the relationship of CDT and the amount of alcohol consumption or presence of liver disease, whereas the influence of alterations in serum transferrin concentrations on CDT has received less attention. In this study, we compared two different methods for measuring CDT (CDTect and %CDT) and total transferrin concentrations in a sample of 83 alcohol abusers (20 patients with alcoholic liver disease and 63 heavy drinkers who were devoid of liver disease, despite excessive alcohol consumption) and 89 controls, who were social drinkers or abstainers. The control population included 53 hospitalized patients with expected abnormalities in serum transferrin concentrations caused by conditions such as negative iron balance, pregnancy, or nonalcoholic liver disease. Both methods gave significantly higher values in alcohol abusers than in controls (p < 0.01), but the overall sensitivity for detecting alcohol abuse was clearly higher for CDTect (59%) than for %CDT (34%). The correlation between the results obtained by the two methods (r = 0.629) significantly improved, when the CDTect values were replaced by the ratio of CDTect/total transferrin (r = 0.770) (p < 0.05). There was a positive correlation between the CDTect and serum transferrin (r = 0.201, p < 0.01), which was significant both in the alcoholics (r = 0.240, p < 0.05), and especially in the controls (r = 0.727, p < 0.001). A significant inverse correlation emerged between %CDT and total transferrin (r = -0.302, p < 0.01). The sensitivities of CDTect and %CDT for correctly classifying alcohol abusers in the subgroup of alcoholic liver disease patients were 90% and 70% and in the subgroup of heavy drinkers without liver disease (49% and 22%), respectively. Specificities for CDTect and %CDT in this sample were 81% and 100%, respectively. However, in the subgroup of hospitalized control patients with abnormal serum transferrin, the specificity of CDTect was only 48%. According to present data, CDTect seems to be more sensitive than %CDT for detecting alcohol abuse. However, any alteration in serum total transferrin concentration markedly decreases the assay specificity. This should be considered when interpreting the assay results in patients with elevated serum transferrin, such as iron deficiency, pregnancy, or liver diseases.

Adult↗
PubMed1996

Screening for problem drinking and counseling by the primary care physician-nurse team.

Present methods to screen for alcohol abuse are generally obtrusive and result in referral to services that deal mainly with alcoholics. These factors deter physicians from identifying alcohol abuse patients at an early stage. In the present study, 81% of all primary care physicians of a single city evaluated (i) the efficiency and the acceptability of a nonobtrusive screening method for the identification of problem drinkers and (ii) the effectiveness of brief cognitive behavioral counseling given by a nurse in a lifestyle context. Patients (n = 15,686) attending the private practices of 42 primary-care physicians were asked four alcohol-neutral trauma questions in the reception area. Physicians asked about alcohol use and alcohol-related problems only to patients with previous trauma. Problem drinkers by defined criteria were offered an appointment with a nurse who, by random assignment, gave either 3-hr of cognitive behavioral counseling over 1 year or simply advised patients to reduce their alcohol intake. The screening method identified 62-85% of expected number of problem drinkers in this population. Following the application of exclusion criteria, 105 problem drinkers were entered in the intervention part of the study. After 1 year, patients who received counseling showed significant reductions in reported alcohol consumption (-70%; p < 0.001), psychosocial problems (-85%; p < 0.001) and serum gamma glutamyl transferase (-32% to -58%; p < 0.02). Physician visits were reduced (-34%; p < 0.02) following counseling. Patients receiving only advice showed neither reductions in psychosocial problems nor in serum gamma glutamyl transferase or physician visits, but reported a 46% reduction (p < 0.01) in alcohol consumption. Data indicate that asking patients about recent trauma is efficient and is well accepted as the first screening instrument in the identification of the problem drinker. Cost of screening per patient is under one dollar. Counseling of 3 hr given by a nurse is markedly superior (p < 0.05) to simple advice in reducing alcohol consumption, objective indicators of alcohol-related morbidity, and the frequency of physician visits.

Adult↗
PubMed1995

Inhibition of gene expression by triple helix formation in hepatoma cells.

The aim of this study was to selectively inhibit human mitochondrial aldehyde dehydrogenase (ALDH2) gene expression by triple helix assembly. Eight 21-mer oligodeoxyribonucleotides were designed to bind to two purine-rich sequences in the 5'-flanking region of the human ALDH2 gene. Gel mobility shift assays showed that triplex formation is sequence-specific for the target duplex and the third strand oligonucleotide. In the presence of Mg2+, but absence of K+, triplex-forming oligonucleotides bind to their target sites with apparent dissociation constants (Kd) in the 10(-7) to 10(-9) M range. Potassium cation virtually suppressed the triplex formation of G-C-rich duplex DNA with natural oligonucleotides, but did not prevent triplex formation with phosphorothioate-modified oligonucleotides. Phosphorothioate-modified oligonucleotides were delivered into human hepatoma Hep G2 cells by cationic liposomes. The reduction in ALDH2 mRNA levels in the cells was determined by the competitive reverse transcription-polymerase chain reaction. One of the phosphorothioate-modified oligonucleotides designed to forma an antiparallel triplex with a target in the 5'-flanking region of human ALDH2 gene (-105 to -125 from the translation initiation codon ATG) reduced by 80-90% the ALDH2 mRNA levels without affecting albumin mRNA levels. Data suggest that triple-helix formation may provide a means to selectively inhibit hepatic ALDH2 gene expression for therapeutic use.

Aldehyde Dehydrogenase↗
PubMed1995

Effect of propylthiouracil treatment on NADPH-cytochrome P450 reductase levels, oxygen consumption and hydroxyl radical formation in liver microsomes from rats fed ethanol or acetone chronically.

The antithyroid drug propylthiouracil (PTU) has been shown previously to reduce hepatic oxygen utilization and to protect the liver from ethanol-induced injury. The present study examined the effect of PTU on hepatic microsomal oxygen consumption and on the activities of NADPH-cytochrome P450 reductase (CYP-reductase) and cytochrome P4502E1 (CYP2E1) in rats receiving ethanol or acetone chronically. Liver microsomes from rats treated with ethanol for 29 days displayed increases in (i) O2 consumption (70%), (ii) hydroxyl radical (.OH) production (49%) and (iii) ethanol oxidation (50%). Microsomal CYP2E1 levels were increased markedly by chronic ethanol administration, while CYP-reductase was affected marginally, but not significantly (P = 0.06). Chronic treatment with acetone for 14 days, produced similar effects, except that .OH production was not enhanced. Administration of PTU (25 mg/kg/day) to ethanol- or acetone-fed rats, for 10 and 14 days, respectively, led to a marked reduction in the levels and activity of CYP-reductase, and to a decrease in the rates of microsomal O2 consumption, .OH production and ethanol oxidation, but did not lower the levels of CYP2E1 or the metabolism of the CYP2E1 substrate N,N-nitrosodimethylamine. These data suggest that the ability of PTU to protect the liver from ethanol-induced injury may be due to a reduction in the levels of CYP-reductase, thereby minimizing the enhancement of microsomal oxygen consumption and free radical generation associated with ethanol-induced CYP2E1 activity.

Acetone↗
PubMed1995

Alcohol consumption by orientals in North America is predicted largely by a single gene.

Orientals consume significantly less alcohol, and show a lower prevalence of alcohol abuse and dependence, than Caucasians. Sociological theories propose that this difference is due mainly to cultural factors. Physiological theories have suggested that the flushing reaction experienced by some Orientals serves as a deterrent to ethanol consumption. The flushing reaction is observed mainly in individuals who possess a mutation in the high-affinity aldehyde dehydrogenase (ALDH2) which renders the enzyme inactive. However, the tendency to flush correlates poorly with alcohol consumption, thus casting doubt on the physiological interpretations. The present study investigates the influence of the ALDH2 allele and of acculturation in North America on alcohol consumption by Orientals born in Canada or the United States. Oriental males carrying the inactive ALDH2(-) allele drink two-thirds less alcohol (6.1 +/- 1.5 vs. 18.2 +/- 2.8 drinks/4 weeks; p < 0.001), show one-third the prevalence of binge drinking (15.2 vs. 42.2%; p < 0.01), and are three times more likely to be abstainers (39.4 vs. 13.3%; p < 0.01) than Oriental ALDH2(+) males carrying the gene for the active enzyme. There were no significant differences in binge drinking or abstinence rates between ALDH2(+) Orientals and Caucasian males. Acculturation in North American society accounted for only 7-11% of the variance in overall consumption (p < 0.02). It is concluded that a single mutation in the high-affinity aldehyde dehydrogenase (ALDH2) gene predicts two-thirds of the alcohol consumption and excessive alcohol use by Oriental males born in North America.

Acculturation↗
PubMed1995

Carbohydrate-deficient transferrin as a marker of alcohol abuse: relationship to alcohol consumption, severity of liver disease, and fibrogenesis.

Carbohydrate-deficient transferrin (CDT) measurements have been widely examined as a marker of excessive alcohol consumption, yet the information on the sensitivity of this method has remained controversial. In addition, little is known of the relationship of this marker and the severity of alcoholic liver disease (ALD). To clarify these issues, we analyzed serum samples from 373 alcohol abusers, including 200 problem drinkers with no apparent liver pathology, 173 patients with clinical or morphological evidence of ALD, and 42 healthy controls. CDT was analyzed by anion-exchange chromatography followed by radioimmunoassay. At a specificity of 100%, the sensitivity of CDT was 36% in problem drinkers reporting a mean of 710 +/- 80 (mean +/- 2SE) g of ethanol/week, as compared with the sensitivities of 44% and 35% for gamma-glutamyltranspeptidase (GGT) and mean corpuscular volume (MCV), respectively. In a subgroup of problem drinkers (n = 51) with the highest ethanol intakes (1160 +/- 180 g of ethanol/week) and severe dependence, the sensitivity of CDT increased to 64%, compared with 55% for GGT and 39% for MCV. In ALD, the CDT values were significantly higher than in the alcoholics with nonliver pathology. However, when such patients were classified according to the clinical, laboratory, and morphological severity of liver disease, CDT was found to be primarily elevated in those with the early stage of ALD, such that there was a significant negative correlation between CDT and the combined morphological index of disease severity (rs = -0.315, p < 0.05). ALD markers of fibrogenesis were elevated more frequently than CDT, showing significant positive correlations with the indices of disease severity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

A simple technique for quantifying intoxication-induced by low doses of ethanol.

A simple technique for the measurement of intoxication induced by low doses of alcohol in the rat was developed. Rats are required to maintained their balance on a rectangular wooden bar that oscillates in a 120 degree angle in an arch-like fashion. A steady baseline can be obtained for each animal with approximately 10 min of training time. Ethanol, in a dose range from 0.5-1.5 g/kg, given orally or by IP route, impairs animal's performance in a dose-related manner. At the same blood ethanol concentration, a higher degree of impairment is observed at higher oscillating frequency. Significant impairment of performance can be detected at ethanol dose of 0.5 g/kg given IP or orally. Pentobarbital and chlordiazepoxide, in doses of comparable potencies to those of ethanol doses also produce a dose-related impairment of performance. The oscillating bar test is a simple but sensitive test that can qualitatively assess intoxication induced by low doses of ethanol or other sedative hypnotic drugs.

Administration, Oral↗
PubMed1994

Long-term treatment of alcoholic liver disease with propylthiouracil. Part 2: Influence of drop-out rates and of continued alcohol consumption in a clinical trial.

Although propylthiouracil has previously been shown to reduce the risk of mortality in alcoholic liver disease by 60%, generalized use of propylthiouracil for this condition has not occurred. Additional data are therefore presented on four aspects to provide a better assessment of its therapeutic effectiveness. First, the characteristics and the prognosis of dropouts were virtually identical in both the drug and placebo groups. Also the methodology and analysis employed, were designed to control for dropouts, thus providing an accurate interpretation of the outcome. Secondly, since 97% of the patients continued to drink, abstinence was not a precondition for the beneficial effect of propylthiouracil. However, the beneficial effect was observed most clearly in those patients who continued to drink at lower levels, whereas lower level drinking per se did not afford protection in placebo patients. Thirdly, serious side effects or clinical hypothyroidism occurred extremely rarely in these patients, many of whom have now received propylthiouracil for over 4 years. Fourthly, we discuss why the outcome in long-term clinical trials in alcoholic liver disease cannot be compared with effects observed in clinical trials lasting only a few weeks. Journal of Hepatology.

Clinical Trials as Topic↗
PubMed1994

Acetate-mediated effects of ethanol.

Ethanol has been shown to increase markedly portal blood flow, primarily by increasing intestinal blood flow. This effect of ethanol is reproduced by acetate, infused at rates equivalent to those leading to endogenous acetate production following ethanol administration. The physiological mediator, adenosine, is also known to increase markedly intestinal and portal tributary blood flow. We have shown that adenosine receptor blockade with 8-phenyltheophylline completely abolishes the effects of ethanol, acetate, and adenosine on intestinal and portal blood flow, suggesting that increases in adenosine tone may constitute a common mechanism mediating the actions of both ethanol and acetate on the splanchnic vasculature. Studies are also presented that show that acetate administration has marked effects on central nervous system function. On two tests, motor coordination and anesthetic potency, both ethanol and acetate showed similar effects. The effects of acetate were fully abolished by 8-phenyltheophylline. The effects of ethanol were partially blocked by 8-phenyltheophylline, with a greater effect of this blocker being seen at low doses of alcohol. Whereas ethanol at low doses increased locomotor activity in mice, acetate markedly reduced it. The effect of acetate on locomotion was fully reversed by the adenosine receptor blocker 8-phenyltheophylline, whereas the activating effect of ethanol on locomotion was markedly enhanced by this blocker. These data suggest that the actions of ethanol on locomotor activity normally result from the combination of a direct stimulatory effect of ethanol per se and an inhibitory effect of acetate, produced endogenously from ethanol. When the latter effect of acetate is abolished by adenosine receptor blockade, the activating effect of ethanol is fully expressed.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde↗
PubMed1994

Ethanol-derived immunoreactive species formed by free radical mechanisms.

Recent studies have shown that the alpha-hydroxyethyl radical (CH3CHOH), a metabolite of ethanol, is produced in vitro and in vivo. We report studies that establish the immunogenicity of alpha-hydroxyethyl radical-derived protein adducts. Rat liver microsomes incubated in the presence of [14C]ethanol and NADPH (under aerobic conditions) incorporate 14C into acid-stable adducts. Incorporation was markedly inhibited by the free-radical scavenger alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone. Rabbits immunized with rat liver microsomes that had been preincubated with ethanol and NADPH generated antibodies that recognized polylysine-acetaldehyde adducts and adducts formed by incubation of proteins with an alpha-hydroxyethyl radical-generating system (ethanol plus H2O2 plus Fe2+). Rabbits immunized with microsomes that had been preincubated with ethanol and NADPH plus alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone generated antibodies that recognized polylysine-acetaldehyde adducts. However, their reactivity against alpha-hydroxyethyl-derived protein epitopes was greatly reduced or was virtually abolished. Data indicate that microsomes metabolizing ethanol generate two types of adducts, acetaldehyde-derived adducts and alpha-hydroxyethyl radical-derived adducts, both of which are immunogenic. Immunization of rabbits with alpha-hydroxyethyl-bovine serum albumin adducts led to the production of antibodies that recognized alpha-hydroxyethyl-rabbit serum albumin adducts but did not recognize the native protein. Chronic alcohol feeding of rats led to the production of antibodies that recognized alpha-hydroxyethyl-rat serum albumin adducts but did not recognize rat serum albumin. The study (i) indicates that alpha-hydroxyethyl radical-derived protein adducts are immunogenic, (ii) supports earlier work that proposed that alpha-hydroxyethyl radicals generated in different systems bind covalently to proteins, and (iii) demonstrates the formation of antibodies to alpha-hydroxyethyl-derived protein adducts after chronic alcohol ingestion in vivo. The findings may have implications in the identification of chronic alcohol abuse and the pathogenesis of alcohol-induced organ damage.

Acetaldehyde↗
PubMed1994

Covalent protein adducts in the liver as a result of ethanol metabolism and lipid peroxidation.

BACKGROUND: The primary mechanisms of ethanol-induced tissue damage have been suggested to include aldehyde-derived protein modifications resulting from ethanol metabolism and lipid peroxidation. Conjugation of reactive aldehydes to a variety of target proteins and cellular constituents have been recently reported. This research was undertaken in order to examine the presence of covalent chemical addition products (adducts) of proteins and acetaldehyde, the first metabolite of ethanol, and those with malondialdehyde, a product of lipid peroxidation, as formed in vivo. EXPERIMENTAL DESIGN: Specific antibodies recognizing acetaldehyde- and malondialdehyde-modified epitopes in proteins were used in immunoperoxidase and double immunofluorescence stainings of liver specimens obtained from ethanol-fed rats and micropigs and from human alcoholics. RESULTS: The centrilobular region of the liver contained the protein modifications both in alcohol-consuming humans and in animals fed ethanol before any apparent histologic damage. With inflammation and fibrosis, such protein modifications were more widespread, and the positive staining for the malondialdehyde-derived modification became more dominant. The presence of the adducts colocalized with the areas of fatty infiltration, focal necrosis and fibrosis. In addition, the erythrocytes of alcohol consumers were found to contain such modifications. CONCLUSIONS: The studies support the view that covalent damage to proteins and cellular constituents induced by aldehyde-derived modifications in vivo may play a role in the sequence of events leading to liver disease in alcohol consumers. Species and dietary differences may be important in the relative contribution of lipid peroxidation to alcohol-induced tissue damage.

Acetaldehyde↗
PubMed1993

Reduction of voluntary alcohol consumption in the rat by transplantation of hypothalamic grafts.

Stimulation of the peripheral renin-angiotensin system has been shown previously to decrease the voluntary intake of ethanol in the rat. The existence of a separate brain renin-angiotensin system, independent from that of the periphery, has been widely demonstrated. The brain renin-angiotensin system plays an important role in the regulation of water and electrolyte balance and neuroendocrine function. However, the role played by this system in the regulation of voluntary alcohol consumption has not yet been studied. The goal of the present work was to assess the feasibility of decreasing the voluntary alcohol intake in a strain of rats (Rapp SS/Jr rats) that have a genetic deficiency responsible for a low activity of the renin-angiotensin system and elevated alcohol intake. Adult Rapp SS/Jr rats received intraventricular transplants of fetal hypothalamic grafts (from normal donors), known to contain angiotensin-immunoreactive cell bodies. Our studies revealed that angiotensin-immunoreactivity in the cell bodies and fibres in the paraventricular, supraoptic and suprachiasmatic nuclei of the hypothalamus in Rapp SS/Jr rats was markedly reduced. Animals that had surviving grafts containing angiotensin-immunoreactive cell bodies in the dorsal third ventricle--but not in the ventral third ventricle, in the lateral ventricles, or sham operated animals--had a 40% decrease of their voluntary alcohol intake, when compared to their intake before surgery, or to the control group. However, water consumption was not reduced in both the sham and transplanted animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Drinking↗
PubMed1993

Polymorphisms of the D4 dopamine receptor alleles in chronic alcoholism.

We have screened genomic DNA for the identification of D4 dopamine receptor polymorphisms. We show that the D4 dopamine receptor genotype in 72 severely affected chronic alcoholics is heterogeneous, with individuals homozygous and heterozygous for the various D4 receptor alleles. Alcoholics demonstrated a greater prevalence of the D4(3) (p < 0.005) and D4(6) (p < 0.005) alleles than has been reported in normals. There was a high prevalence of nicotine abuse among all D4 genotypes. The frequency of other drug abuse was higher in the D4(3,3) and the D4(4,7) groups, and the family history was strongly positive in the D4(2,4) group. The distribution of the D2 alleles showed equivalence in all D4 genotypes, except in D4(4,6) and D4(4,7) in whom the prevalence of the D2 A1A2 allele was 2-fold higher. The polymorphic variations of the D4 receptor genes should be among the factors considered in the assessment of individual differences in susceptibility to disorders such as alcohol abuse or drug addiction that may be mediated through central dopaminergic systems.

Adult↗
PubMed1993

Simple method for the preparation of antigen emulsions for immunization.

We have developed a rapid, safe, and reliable method to prepare emulsions of water-soluble antigens in an adjuvant oil phase for immunization purposes. The method, based on well established emulsification principles, employs a three-way 'T' connector to which three disposable syringes are attached. The system allows the stepwise addition of small volumes of the water phase, into the oil phase. We have compared the time required for emulsification, the rate of antigen release from the emulsion into a physiological phase, and the immunogenic properties of bovine serum albumin and transferrin contained in emulsions made by the new stepwise addition method, with those made by the widely used double-hubbed needle method. We report a significantly shorter (P < 0.001) and a more reproducible emulsification time for the stepwise addition method (6.1 +/- 2.1 min; mean +/- SD) than for the double-hubbed needle method (41.1 +/- 28.0 min). The stepwise addition method always yielded water-in-oil emulsions, while the double-hubbed needle method failed, about 20% of the time, to produce a water-in-oil emulsion after 120 min of mixing. Since the stepwise addition method employs a connector with a larger inner diameter (1.75 mm) than the one required for the double-hubbed needle method (0.84 mm); the pressure required for the former is markedly reduced compared with that required for the latter, thus making the new method safer and less labor-intensive. The rate of antigen release from the emulsions was significantly slower when the stepwise addition method was employed (P < 0.01). There were no differences in viscosity and stability in the emulsions prepared by the two methods. The ability of antigen-containing emulsions to elicit an immune response was found to be identical by the two methods; no significant differences were found in antibody titers as determined by enzyme-linked immunosorbent assays. These characteristics make the stepwise addition system the method of choice.

Adjuvants, Immunologic↗