1182

BIOCHEMICAL SOCIETY TRANSACTIONS

The nature of apolipoprotein B in rat chyle TAYFUN GULDURand PETER A. MAYES Ilivision o f Biochemistry, Department of Veterinary Basic Sciences, Royal Veterinary Cbllege, London N WI OTU, U.K.

EDTA, 0.02% (w/v) NaN, and ampicillin (0.05 mg/ml). These preservatives were used at these concentrations in all subsequent manipulations of lipoproteins. In half of the chyle collections and their subsequent processing, the proteinase Apolipoprotein B (apo B) is the integral apolipoprotein of inhibitors &-amino caproic acid (10 mM) and leupeptin ( 1 chylomicrons, very-low-density lipoproteins (VLDL), mM) were also added. intermediate-density and low-density lipoproteins. It is Two millilitres of chyle were layered under NaCl ( d l .006) heterogeneous, occurring in two forms. In humans, the larger in 6.5 ml cellulose tubes and centrifuged in a fixed-angle apo B-100 is formed by the liver in VLDL and the smaller rotor (MFT-50.6) of an MSE Europa 55 ultracentrifuge for apo B-48 is produced by the intestines in chylomicrons. In 6 x 10' gmin at 4°C. The top 1 ml fraction containing large the rat, apo B-48 is also found in liver VLDL. Lee et ul. [ 1 , 2 ] chylomicrons ( > 100 nm) were collected and subjected to reported small amounts of apo B-100 in rat chyle which two further centrifugations under the same conditions. At disappeared on storage o r repeated Centrifugation. They each stage the lipoproteins were analysed by SDS/PAGE [4]. suggested that proteinases, present in chylomicron prepara- In Fig. 1, gel A from crude chyle shows a heavy band corretions, were responsible for degradation of apo B-100 into sponding to apo B-48 and a lesser band for apo €3-100. apo B-48. However, it has also been shown that apo B-48 Repeated centrifugation led to progressive loss of the apo has thc same amino acid sequence as the N-terminal region B-100 band, but retention of the apo B-48 band (gels B, C, o f apo B- I00 and is formed from the same mRNA in intes- D). In some initial centrifugations (gel B) the apo B- 100 band tine, apo €3-48 being released as a result of the action of a cannot be detected but in others (gcls E and F ) it remains premature stop codon [ 3 ] .The nature of the origin of apo just visible. These results confirm the findings of Lee et ul. B-48 in chyle is of sufficient importance to warrant re- [ I ] . However, we could find no evidence of proteinase investigation of the significance o f proteinase activity in activity over the short term, as gels E and F demonstrate. generating this apoprotein. The infranatant from the initial centrifugation was recentriMalc Wistar rats (340-350 g) were tube fed 1.5 ml of corn fuged at d1.006 for 5.7 x l o 7 gmin. Gel G from the resulting oil fortified with 10 i.u. o f a-tocopheryl acetate/ml as anti- supernatant fraction, representing small chylomicrons and oxidant. After 1 h they were anaesthetized with sodium VLDL, contained distinct apo B-100 as well as apo B-48 pcntobarbital (60 mg/kg body weight) and the thoracic duct bands. Further centrifugation of the infranatant from this cannulatcd with polycthylene tubing. Chyle was collected in latter centrifugation at d l .O 19 also showed the presence of an ice bath in the presencc of final concentrations of 3 mM- apo B- 100 as well as apo B-48 (gel I). Thus, our results d o not support the hypothesis that apo Abbreviations used: apo 13. apolipoprotein B; VLDL. very-low- B-48 in rat chylomicrons is formed by proteolytic degradadensity lipoproteins. tion of apo B- 100. Apo B- 100 seems to be stable even after

A

B

C

D

E

F

I

Molecular mass

Wa)

Apo B-100

Apo 8-48

205

116

97

Fig. I . Apolil,c)i~roteiricwnposition o f rut chyle fructions reailtirig frorn seqiicntiul cwitrifiigutiori ut vurioirs densities Lipoprotein fractions were delipidated and thc apolipoprotcins separated by PAGE in the prescncc of SDS using a 3.5'%,(w/v) gel. A. crude chyle; €3, E. F, supernatant from centrifuging crude chyle for 6 x 1Oi gmin (d1.006);C, supernatant from ccntrifuging B for 6 x 1 Oi gmin (dl ,006); D. supernatant from centrifuging C for 0 x 1O 5 gmin ( t l l . 0 0 6 ) ;G. supernatant from centrifuging the infranatant from F for S.7 x 107 gmin (dl .006); H. protein standards; 1. supernatant from centrifuging the infranatant o f G for 5.7 x 1O7 gmin ( d l .O 19). Proteinase inhibitors were present throughout the preparation o f A. 13. F and G. but absent from E and 1.

635th MEETING, ABERDEEN

1183 The skilled technical assistance o f Michael Avella and thc support of the Turkish Higher Education Committee and the British Heart Foundation are appreciated.

three centrifugations (gel I). Rather, we believe that successive centrifugation of large chylomicrons for 6 x 10' gmin results in their purification with loss of VLDL and small chylomicrons containing apo B- 100. The results demonstrate that purified large chylomicrons contain apo B-48, but do not contain apo B- 100 and that the apo B-1 00 in chyle is located in the small chylomicron and VLDL fraction. As remnants derived from apo B- 100-containing lipoproteins ultimately form atherogenic low-density lipoproteins, the results imply that large chylomicrons, which lack this apoprotein, are not as potentially atherogenic as small chylomicrons or VLDL containing apo B- 100.

I . Lee. D. M.. Koren. E., Singh. S. & Mok. T. ( 1 984) Hiochem. Biophys. Hes. ('ommiin. 123, I 140- I 156 2. Lee. I). M. & Singh. S. (1088) Hiochim. Hiophys. Acrri 960. 148-156

3. Powell. L. M., Wallis. S. C., Pease. R. J., Edwards, Y. H., Knott, T. J. & Scott. J. ( 1087) C'ell(('umhridge,Mrrss.) 50. 83 1-840 4. Laemmli, U. K. ( 1070) Nutitrc (Lotidon) 227, 680-685 Received I8 June 1990

Free radical activity during percutaneous trans-luminal coronary angioplasty JOHN R. PATERSON,* KEITH G. OLDROY D.t ALAN G. RUMLEY,$ HANY ETEIBA,t ALAN P. RAE,t IAN HUTTONt and STUART M. COBBEt *Iti.siitiiie of' Riochemi.siry arid t Depurtmetii of'h4edicul Curdiology, Royal Infirmary, 1 0 Alexandra I'urude, Glusgow (;4 O.SF,U.K . utid $Depurimetii of I'uihologicul Riochetnistry, Gurtriuvel General Ilospiiul, Glusgow GI2 OYN, U.K.

tion, simultaneous blood samples were taken from the aorta and coronary sinus before PTCA and at 2 (coronary sinus only; n = 7) and 10 min after each series of balloon inflations. The stenoses, which were all initially successfully dilated, included ten lesions in the left anterior descending and three in the left circumflex coronary arteries. The patient with unstable angina sustained an acute occlusion 2 h after the procedure. The mean number of balloon inflations was 3.2 per patient (range 2 to 7) and the mean total duration of inflations was 162 s (range 00-360). Two markers of free radical activity, the plasma dienc conjugate o f linoleic acid (18:2) [Y, 1 1 I and its ratio to the unconjugated diene ( 1 X:2) [Y, I21 and plasma lipid peroxides were determined. The former were measured by an h.p.1.c. method [ 101. Plasma (EDTA) lipid peroxide concentrations were measured using a modified h.p.1.c. method [ 1 1 I. Plasma ( 100 p l ) was added t o phosphoric acid ( 1.5 ml, 0.44 M ) , and thiobarbituric acid ( 0 . 5 ml, 42 mM) in a polypropylene tube. The mixturc was placed in boiling water for 1 h, and then in ice for 5 min. Ethyl acetate/butanol ( 1.5 ml; 2: 1, v/v) was added t o the tubes and the contents mixed for 5 min on a rotary mixer. The tubes were then centrifuged (3000 rev./ min for S min) and 1 ml o f the organic layer removed and evaporated to dryness at 40°C under oxygen-free nitrogen. The residue was rc-dissolved in mobile phase ( 1 S O pi) and SO pI injected on the h.p.1.c. column. The mean percentage molar ratio (MR) of the 9 : 11 to 9:12 isomers of linoleic acid and concentration o f lipid peroxides (LP) before, and 2 and 10 min after PTCA are given in Table 1. There was no increase in either the percentage MR of the 9 : 1 1 to Y: 12 isomers of linoleic acid or in LP measured at 2 and 1 0 min after PTCA. Nor was there any difference in these markers of free radical activity between samples taken from the aorta (blood entering the coronary arteries) and from the coronary sinus (blood leaving the

Evidence that oxygen-derived free radicals are generated following myocardial ischaemia and reperfusion comes mainly from animal work 11 -31. However, the results of such studies have provided conflicting results [ 4.5I. perhaps due t o differences in the ischaemic models and radical-scavenging agents used. A few limited studies have been carried out in patients with myocardial infarction [ 6, 7 I, undergoing aorto-coronary bypass grafting [ 81 or during ischaemia induced by pacing [ 91. Oxygen-derived free radicals were believed to have been generated, although in most studies only one marker o f free radical activity was determined and assays used wcrc relatively non-specific. Increased intra-cardiac frec radical activity could contribute t o endothelial injury and. if generated following percutaneous trans-luminal coronary angioplasty (PTCA). might increase the risk of rcstcnosis. The aim o f this study was to measure free radical activity using two specific h.p.1.c. assays before and immediately after PTCA and t o correlate this activity with the degree of ischaemia. Ten patients (mean age 56.2 years, eight males) undergoing single vessel PTCA were studied. One subject had unstable angina, the remainder all had stable angina with positivc exercise tests. Drug therapy in the patients included P-blockers (ti = 7), calcium antagonists (ti = 8), nitrates (ti = 0 ) and aspirin (ti = 10). After full systemic hcparinizaAhhrcviationx used: P'I'CA, percutaneou\ trans-luminal coronary angioplasty; MR, molar ratio; LP, lipid peroxides.

Table 1 . 1.i.C.eriidicul uctiviiy before und immediately ufler I'TCil Blood samples were taken from the aorta and coronary sinus before. and 2 and 10 min after PTCA. Free radical activity was measured as the percentage M R of 9 : I 1 to 9: 12 isomers of linoleic acid and LP. Values shown are means f s.D., n = 10 for before and 1 0 min after PTCA. and ti = 7 for 2 min after PTCA. Before PTCA

2 min after PTCA

10 min after

PTCA

MR (%)

LP ( p M )

MR (%)

LP ( I i M )

M K (%)

1,P (,uM)

2.9 ( 1 3 ) 3.0 ( 1.4)

2.4 ( 1 . O )

-

-

2.Y (1.4)

2.8 (1.2)

2.5 ( 1 . 1 )

3.0 ( 1 .8) 3.0 (1.7)

2.6 ( 1.2) 2.6 (0.8)

~~

Aorta

Coronary \inu\

VOl. I 8

The nature of apolipoprotein B in rat chyle.

1182 BIOCHEMICAL SOCIETY TRANSACTIONS The nature of apolipoprotein B in rat chyle TAYFUN GULDURand PETER A. MAYES Ilivision o f Biochemistry, Depart...
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