April 1995 AASLD A1093

RECOMBINANT HEPATITIS B VACCINE-LONG TERM EF- F ICACY IN HOSPITAL PERSONNEL.Nayana Joshi, Y.R.NAGARJUNA KUMAR D.V.Sr in ivas,Aj i t Kumar, P.N.Rao.Department of Gastroenterology, Nizams Institute of Medical Sciences,Hyderabad,INDIA The data regarding im- mune response and long term follow up of Hepatitis B vac- cination is scanty from India.One hundred susceptible healthy hospital personnel of a tertiary care centre who were hepatitis surface antigen (HBsAg) and antibody(antiHBs) negative were vaccinated with 20/agms of recombinant yeast derived vaccine(Engerix B) at 0,1 & 6 months interval in 1988- 8g.of the 81 persons who completed the schedule, a pro- tective antibody (antiHBs) titre of)10mlU/ml is achieved in 96%(78) with a mean antibody titre of 264.6±194.TmlU/ml.65 vaccinees (Male=44 Female=21, aged 21-50yrs) of the 78 who had a protective antibody titre were followedup and at the end of 5yrs(1994),20%(13) of the vaccinees were hav- ing antibody titres less than 10mlU/ml. Protective antibody titres were seen in 80% (n=52) of the vaccinees with a mean titre of 116.4*103.1mlU/ml.ln males,the mean antibody titres have fallen from 258.78±189.92 to 78.39±75.81 mlU/ml com- pared to the females in whom the titres have fallen from 286.48.+.201.84 to 148.23.+,136mlU/ml.ln the age group of 21-30 yrs(n=35)the mean titre has fallen from 274.2 -~166.2 to 130.4±95.7mlU/ml and in the age group of 31-40 yrs(n=27) the mean t i t res have fal len from 272.7-.234.7 to 103.2~114.5mlU/ml. Loss of protective antibody is seen in 11%(4/35) of 21-30 yr age group compared to 29%(8/27) in the age group of 31-40 yrs. None of the vaccinees devel- oped Hepatitis B surface antigen or clinical hepatitis in the follow up period. We conclude that in the long term followup 1) ant ibody titres are inf luenced by age and sex 2) 80% maintain protective antibody titre after successful primary vaccination.

COMPARISON OF RAPID ULTRAFILTRATION AND GELCHROMATOGRAPHY OF BILIARY LIPIDS IN HEPATIC AND GALLBLADDER BILE. D. Jfings__t, C.E. Schriever. Department of Medicine II, Klinikum Grosshadern, Ludwig-Maximilians-University of Munich, Germany.

It has been shown by high resolution chromatography, ultra- centrifugation, electron microscopy and laserlight scattering that cholesterol-phosphotipid vesicles (diameter: 60-80nm) and mixed micelles of bile acids, phospholipids and cholesterol (diameter: 5-6ran) are present in human bile. Since all above mentioned methods to characterize these lipid aggregates in human bile are prone to artifacts, lipid transport in bile is still incompletely understood. We, therefore, used rapid ultrafiltration to study the characteristics of lipid aggregates in biIe and compared the results with convenient gelchromatography. After ultracentrifugation of hepatic biles [n=13; total lipid concentration (TLC): 1.3 ± 0,3 g/dl], depending on the pore Size (PS) of the membranes (220, 100 and 50 nm) the mean percentage of ultrafiltrable bile acids (BA) decreased considerably (Fig.). Nearly identical results were obtained for the mean percentage of ultrafiltrable phospholipids (PL) and cholesterol (C). Surprisingly, a similar ultrafiltration characteristic of bitiary lipids was found in more concentrated gallbladder biles (o=12; TLC: 7.8 ÷ 0.6 g/dl).

100 Hepat ic B i le 100 Gal lb ladder B i le o gO - S0

60 ~ 60

,to ~- 4~ • ~ -~ 20

B A PL C BA PL C B A PL C ~ ~ BA PL C BA PL C BA PL C

PS: 220 nm PS: t00 nm PS: 50 nm ~ PS: 220 nm PS: 100 nm PS: 50 nrn

The percentage of ultrafiltrable lipids was not related to the distribution of bile lipids in vesicles and mixed micelles after gelchromatography or to the concentrations of BA, PL, C or cholesterol saturation. Conclusion: Our ultrafiltration studies suggest that large molecular aggregates of C, PL and BA are the major transport form of these lipids in hepatic as well as in gallbladder bile.

CRYSTALLIZATION OF CHOLESTEROL: EFFECT ON THE RELATION OF CHOLESTEROL IN VESICLES AND MICELLES IN GALLBLADDER BILE OF PATIENTS WITH CHOLESTEROL STONES. D. Jtingst, G. Meyer*, C. yon Ritter. Department of Medicine II and Surgery*, Klinikum Grosshadem, Ludwig-Maximilians-University of Munich, Germany.

Biliary cholesterol is thought to nucleate primarily from phospholipid vesicles and the close association of vesicles and crystals in human gallbladder bile supports this contention. Transition of cholesterol from micelles to vesicles seemed to be of key importance in this process. We, therefore, studied the effect of cholesterol crystallization on the relation of vesicular to micellar cholesterol in gallbladder biles.

Gallbladder bile samples were obtained at laparoscopic surgery from 12 patients with cholesterol gallstones. After ultracentrifugation crystal-free bile was divided in aliquots, placed in sterile tubes, flushed with N2, sealed and incubated at 37~C. Separation of vesicular and micellar cholesterol was performed by gelchromatography of 0.5ml bile on Sephacryl-S-300 superfine columns with an eluting buffer containing 10mM sodium cholate. The samples were analyzed at zero time, at the first appearance of cholesterol crystals and 10 days thereafter. Nucleation of cholesterol crystals occurred within 1-i 1 days (median 2 days) and during the total observation period of up to 21 days the percentage of cholesterol of total biliary lipids decreased slightly from (11.8 ± 3.5 to ll.0 ±1.6 and 10.2 =L 1.9 mmol/1; mean ± SEM) and the cholesterol saturation index from 1.66 ± 0.37 to 1.64 ± 0.22 and 1.33 ± 0.23. Vesicular cholesterol remained between 21.5 ± 2.7%, 25.7 ± 3.4% and 22.5 ± 2.6% of total biliary cholesterol and cholesterol of mixed micelles varied between 78.1 ± 3.7%, 74.0 ± 4.5% and 77.1 =t= 3.7%. Conclusion: During the in-vitro nucleation of biliary cholesterol only minor nonsignificant changes of cholesterol concentration and distribution of cholesterol between the different carriers were observed. An explanation for this I"mding is that the amount of newly formed cholesterol crystals is of minor quantitative importance and, therefore, did not affect the micelle-vesicle relation of cholesterol significantly.

This study was supported by the Else-Kroner-Fresenius Stiffung

PROTEIN-LIPID INTERACTION: EFFECT ON THE DISTRIBUTION OF CHOLESTEROL IN VESICLES AND MICELLES IN GALLBLADDER BILE. D. Jtingst, R. del Pozo. Department of Medicine II, Klinikum Grosshadern, Ludwig-Maximilians-University of Munich, Germany.

Cholesterol is transported by low molecular mixed micelles and high molecular phospholipid vesicles in gallbladder bile. Biliary proteins seem to modulate the distribution of cholesterol between these carriers. To further elucidate the underlying mechanism we studied the significance of protein-lipid interaction in gallbladder bile. Biliary lipids and proteins were separated using gel filtration chromatography (Sepharose 2B, 30 x 1.5 cm). After elution with 10mM Na-cholate buffer bile acids, phospholipids, cholesterol and proteins were measured in 1.0 ml fractions. Model bile free of protein (cholesterol saturation index: 1.0; total lipid concentration: 9.8 g/dl) was used in control experiments. To generate vesicles bile samples were dialyzed to remove the bile acids and subsequently fractionated on Sepharose 2B colmrms without the addit~p of Na-cholate in the elution buffer. Protein-lipid interaction was proven by rechromatography and 10% KBr density gradient ultracentrifugation (vertical rotor Sorvall TV- 865; 300.000g x 6 h) of pooled and concentrated fractions. More than 90% of biliary proteins coeluted with mixed micelles in gallbladder bile of patients with pigment, mixed or cholesterol stones. After dialysis of the bile samples and gel filtration a partial transition of mixed micelles to vesicles was observed in biles from patients with mixed and cholesterol stones but not seen in bile from patients with pigment stones. A strong association between biliary proteins and low molecular phospholipid-cholesterol aggregates counteracts the formation of vesicles, which was complete in protein free model biles after dialysis. Conclusion: Protein-lipid interaction seems to be of great physiological importance for biliary cholesterol transport and, therefore, may play a crucial role in the pathogenesis of cholesterol gallstones.

This study was supported by the Else-Kr6ner-Fresenius Stiftung.