Correlation between restriction fragment length ...

62 1 st MEETING, LONDON peroxymethyl uracil, and all three considerations might contribute to the much lower enzymic rates obtained with the DNA hydroperoxide. DNA hydroperoxides are mutagenic (Thomas et al., 1976) and may be one of the factors responsible for mutagenicity of X- and y-irradiation or the carcinogenicity in the rat of a choline- and methionine-deficient diet which is associated with lipid peroxidation in the nucleus (Rushmore et a[., 1984; Tan et al., 1986) and strand breaks in nuclear DNA (Rushmore et al., 1986). The reduction of these hydroperoxides should be an important step in the limitation of their potential for nuclear DNA damage. Removal of the resulting reduction products, 5-hydroxymethyl uracil or thymine glycol, by appropriate DNA glycosylases are further steps which should allow complete repair of the DNA to occur (Hollstein et a[., 1984; Ames, 1986). If such a repair mechanism were to operate it is essential that peroxidized DNA is accessible to GSH transferases. In the case of nuclear DNA, GSH transferases or Se-dependent GSH peroxidase would of necessity be chromatin proteins similar to other repair enzymes. In this respect it may be significant that the non-histone chromatin protein BA is composed of GSH transferase subunits 3 and 4 (Bennett et al., 1986). Whereas in the rat liver, Se-dependent GSH peroxidase activity is much greater than Se-independent GSH peroxidase activity is due to GSH transferases, the opposite is true in the human, while in the guinea pig the Se-dependent enzyme appears to be absent (Lawrence & Burk, 1978). This suggests that these two forms of GSH peroxidase may act similarly with respect to the reduction of organic hydroperoxides in vivo.

629 A h , P., Mannervik, B. & Jornvall, H. (1985) FEBS Lett. 182,319-322 Ames, B. N. (1986) in Antimutagenesis and Anticarcinogenesis Mechanisms (Shankel, D. M., Hartman, P. E., Kada, T. & Hollaender, A., eds.), pp. 7-39, Plenum Press, New York, London Beale, D., Meyer, D. J., Taylor, J. B. & Ketterer, B. (1983) Eur. J . Biochem. 137, 125-129 Bennett, C. F., Spector, D. L. & Yeoman, L. C. (1986) J. Cell B i d . 102, 600-609 Buege, J. A. & Aust, S. D. (1978) Methods in Enzymol. 52, 306-310 Daniels, M., Scholes, G., Weiss, J. & Wheeler, C. M. (1957) J. Chem. Soc. 200-239 Hollstein, M. C., Brooks, P., Linn, S. & Ames, B. N. (1984) Proc. Natl. Acad. Sci. U . S . A . 81, 5633-5637 Lawrence, R. A. & Burk, R. F. (1978) J. Nutr. 108, 21 1-215 Meyer, D. J., Beale, B., Tan, K. H., Coles, B. & Ketterer, B. (1985) FEBS Lert. 184, 139-143 Prohaska, J. R. & Ganther, H. E. (1976) J. Neurochem. 27, 1379-1387 Rushmore, T. H., Lin, Y. P., Farber, E. & Ghoshal, A. K. (1984) Cancer Let/. 24, 251-255 Rushmore, T. H., Farber, E., Ghoshal, A. K., Parodi, S., Pala, M. & Taningher, M. (1986) Carcinogenesis 7 , 1677-1680 Schulte-Frohlinde, D. & von Sonntag, C. (1985) in: Oxidative Stress (Sies, H., ed.), pp. 11-36, Academic Press, London Tan, K. H., Meyer, D. J., Coles, B. & Ketterer, B. (1986) FEBS Lett. 207, 231-233 Tan. K. H., Meyer, D. J. & Ketterer, B. (1986) Free Radical Res. Commun. in the press Thomas, H. F., Herriott, R. M., Hahn, B. S. & Wang, S. Y. (1976) Nuture (London) 259, 341 -342 Vander Jagt, D. L., Hunsaker, L. A,, Garcin, K. B. & Royer, R. E. (1985) J . Biol. Chem. 260, I 1603- I I6 10

Received 8 December 1986

Correlation between restriction fragment length polymorphisms of apolipoprotein B and serum lipid levels in patients with peripheral and coronary arterial diseases M. V. MONSALVE,* P. J . TALMUD,* A. KAY,* S. WISEMAN,? J . POWELL,? R. GREENHALGH,? and S. E. HUMPHRIES* *Charing Cross Sunley Research Centre, Lurgan Avenue, London W6 8 L W , U . K . and ?Department of Surgery, Charing Cross Hospital, Hammersmith, London W6 XRF, U . K . There is now considerable evidence that alterations in the level or functions of some of the apolipoproteins are important factors in the development of hyperlipidaemia and atherosclerosis. Several studies have shown a positive correlation between elevated plasma concentrations of lowdensity lipoprotein (LDL), cholesterol and premature coronary heart disease (Whayne et al., 1981, Brown & Goldstein, 1983). Apolipoprotein B (apo B) is an important constituent of the triglyceride-rich very-low-density lipoprotein and is the major protein of the cholesterol-rich LDL. Apo B is made by the liver and it is the ligand responsible for the receptor-mediated uptake and removal of LDL from the circulation. It is one of the largest monomeric protein known with a calculated M , of 512.937. The gene for apo B has been assigned to the short arm of the chromosome 2 (p23 -+ pter). DNA probes for the human apo B gene have recently been isolated (Carlsson et al., 1985; Knott et al., 1985; Lusis et al., 1985; Shoulders et al., 1985) and these have been used to detect a number of common restriction fragment length polymorphisms (RFLPs) of the gene (Shoulders et al., 1985; Abbreviations used: LDL. low-density lipoprotein; apo B, apolipoprotein B; RFLP. restriction fragment length polymorphism.

Vol. 15

Priestley et al., 1985; Talmud et al., 1985; Barni et a[., 1986). There are three common RFLPs of the apo B gene detected with the enzymes EcoRI, MspI and Xbai. It has been recently reported that variations in the gene for apo B are associated with the determination of serum cholesterol in the normolipidaemic population (Law et a[., 1986; P. J. Talmud, N. Barni, A. M. Kessling, P. Carlsson, C. Darnfor, G. Bjursell, D. Galton, V. Wynn & S. E. Humphries, unpublished work). Within the normolipidaemic population, homozygotes for the XZ allele had a significantly higher serum cholesterol than homozygotes for the X I allele, with individuals of the genotype X I X Z having an intermediate value. In addition, for the RFLP detected with XbaI, the frequency of one of the alleles X 2 , is significantly higher in the group of patients with type 111 hyperlipidaemia (P. J . Talmud, N. Barni, A. M. Kessling, P. Carlsson, C. Darnfor, G. Bjursell, D. Galton, V. Wynn & S. E. Humphries, unpublished work). We have obtained blood samples from 100 patients with documented peripheral arterial disease (stroke or intermittent claudication) who attend the Department of Surgery of Charing Cross Hospital (London). Samples were taken after 12 h fasting. Serum triglycerides and cholesterol were enzymically determined using test kits from BoehringerMannheim. Apo B was measured by end-point laser nephelometry. For these individuals we have determined the genotype for the polymorphism of the apo B gene detected with the enzyme XbaI. DNA preparation, restriction endonuclease digestion, Southern blotting, hybridization to [32P]apoB and autoradiography were as those previously described by Kessling et al. (1985), Talmud et al. (1985), Barni et al. (1 986).

630

BIOCHEMICAL SOCIETY TRANSACTIONS Table 1. Genotype distribution and relative allele frequency for apo B XbaI RFLP in controls, all patients and normo- and hyper-lipidaemic patients Statistical significance: * P < 0.01, t P > 0.5. Allele frequency

Genotype II

12

22

Total

I

2

Controls

30

76

33

139

0.51

0.49

All patients

31

62

7

I00

0.62

0.33

Normolipidaemic patients H yperlipidaemic patients

15

25

2

42

0.655

0.345

16

37

5

58

0.595

0.405

xz 12.55*

I .02t

Genotype and allele frequencies for the apo B XbaI RFLP are presented in Table 1. The frequency of the XbaI cutting site ( X 2 allele) is significantly lower in the patient group compared with the clinically well and normolipidaemic London population ( P < 0.001). The frequency of the XbaI was determined for the subgroup of the normo- and hyper-lipidaemic patients. There was no significant difference in the allele frequency between these two patient groups tested. In the sub-group comprising normolipidaemic patients ( n = 42), individuals with the genotype XbIXbI have a lower mean level of total serum cholesterol and apo B compared with individuals with the genotype Xb2Xb2, with individuals of the genotype XbIXb2 having intermediate values (Fig. I). For this sample, it is possible to estimate the contribution to the level of serum cholesterol associated with the XbaI alleles of the apo B gene, a statistical analysis using the formula described by Sing & Davignon (1985). We estimate that on average, the effect associated with the X I allele is to lower cholesterol levels by 0.097 mmol/l, while the effect of the X 2 allele is to raise cholesterol levels by 0.168 mmol/l. These data, from a group of patients, support previous results that genetic variation associated with the gene for apo B is involved-in determining the level of serum

cholesterol and apo B. The observation that the frequency of the XbaI RFLP is different in the patient group suggests that variation in the gene for apo B is involved in predisposing an individual to atherosclerosis. This appears to be independent of the involvement of this gene in the development of hyperlipidaemia. It is known that the single base-pair change which gives rise to the XbaI RFLP does not alter the coding sequence of the gene (Carlsson et al., 1986). A possible explanation for the relationship of the XbaI DNA polymorphism with lipid levels is that the XbaI RFLP is in population association with some other functionally significant change in the coding region of the gene. Changes in amino acid sequence may, for example, alter the affinity of the apo B for the LDL-receptor, and result in an altered clearance rate of LDL from the serum. Alternatively, differences in the rate of production of the apo B protein could result from changes in the promotor region of the apo B gene. Inferences from these results must be tentative, because of the small sample size. However, the observation that the frequency of the XbaI RFLP is different in the patient group suggests that the apo B locus may be one of the genes that is involved in the predisposition of an individual to develop atherosclerosis. We gratefully acknowledge financial support from the British Heart Foundation and the Charing Cross Sunley Research Trust. A.K. was the recipient of a Nuffield Foundation award.

.

c 2.0 M

v

E

u

1.5

1 .o I

I

Fig. 1. Lipid levels in the normolipidaemicpatients with diferent XbaI genotypes

The mean plus 95% confidence limits are shown for serum cholesterol ( a ) and triglyceride ( b ) and apo B levels (c) for individuals with the genotype X I X I , X I X 2 and X2X2. The respective means and standard deviations of cholesterol levels (mmol/l) for the three genotype were 5.33 (k1.07), 5.62 (k0.99), 6.35 ( * 0.38), of the triglyceride levels (mmol/l) were 1.52(?0.388), 1.36(+0.436), 1.50(f0.66),andoftheapo B levels (g/l) were 1.37 (&0.228), 1.46 (*0.20), 1.55 (*0.003), 1.89 ( & 0.000).

Barni, N., Talmud, P. J., Carlsson, P.. Azoulay, M., Darnfors, C., Harding, D., Weil. D., Grzeschik. K. H., Bjursell. G., Junien, C.. Williamson, R. & Humphries (1986) Human Genet 73, 313-319 Brown, M . S. & Goldstein, J. L. (1983) in The Meraholic Basis qf Inherited Disease (Stanbury, J. B., Wyngaarden, J . B., Frederickson, D. S., Goldstein, J. L. & Brown, M. S. eds.), pp. 500 550, MacGrawHill, New York Carlsson, P., Olofsson, S. O., Bondjers, G., Darnfors, C., Wiklund, 0. & Bjursell, G . (1985) Nucleic Acid Res. 13, 8813-8824 Carlsson, P., Darnfors, C., Olofsson, S. 0.& Bjursell, G., (1986) Gene in the press Kessling, A. M., Horsthemke, B. & Humphries, S. E. (1985) Clin. Genet. 28. 296-306 Knott, T. J., Rall, S. C., Innerarity, T. L., Jacobson, S. F., Urdea, M. S., Levy-Wilson, B., Powell, L. M., Pearse, R. J., Eddy, R., Nakai, H., Byers, M., Priestly, L. M., Robertson, E., Ball, L. B., Betshalz, C., Shows, T. B., Mahley, R. W. & Scott, J. (1985) Science 230, 37 4 3 Law, A., Wallis, S. C., Powell, L. M., Pease, R. J., Brunt, H., Priestley, L. M., Knott, T. J., Scott, J., Altman, D. G . , Miller, G . J., Rajput, J. & Miller, N. E. (1986) Lancet i, 1302-1303 Lusis, A. J., West, R., Mehrabian, M., Reuben, M . A,, LeBoeuf, R. C., Kaptein, J. S., Johnson, D. F.. Schumaker, V. N.. Yuhasz, M. P., Schotz, M. C. & Elovson, J. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 45974601 Priestley, L., Knott, T., Wallis, S., Powell, L.. Pearse, R., Simon, A. & Scott, J. (1985) Nucleic Acid Res. 13, 6789-6794

1987

621st MEETING, LONDON Shoulders, C. C., Myant, M. B.. Sidoli, A,, Rodriguez, J. C., Cortese, C. & Baralle. F. E. (1985) Atherosclerosis 58, 277-292 Sing, C. F. & Davigrjon, J. (1985) Hum. Genet. 37, 268-285 Talmud, P. J., Azoulay. M., Junien, C., Carlsson, P., Weil, D., Grzeschik. H. K., Bjursell, G.. Williamson, R. & Humphries, S. (1985) Cyrogenel. Cell Gene! 40,759

63 1 Whayne, T. F., Alaupovic, P., Curry, M. D., Lee, E. T., Anderson, P. S., Schechter, E. (1984) Atherosclerosis 39, 41 1 4 2 4

Received 1 December 1986

Alterations in leucocyte prostaglandin production in ulcerative procto-colitis NEVILLE A. PUNCHARD,* RICHARD B. HUBBARD,* JOHN CASON,* ALEXANDER T. GREEN,* ROBIN A. WOLSTENCROFTt and RICHARD P. H. THOMPSON* *Gastrointestinal Laboratory and t Immunology Department. Rayne Institute, St. Thomas’ Hospital, London SEI 7EH, U . K . Ulcerative procto-colitis (UC) is a chronic inflammatory disease of unknown aetiology that affects the rectum and colon. Features of active disease include disturbed function and acute-phase responses. The essential fatty acid derivatives prostaglandins (PG) may be involved in the pathology of UC and in an active disease increased levels have been reported in stools, rectal dialysis fluid, mucosal biopsy specimens and urine (Hawkey & Rampton, 1985). However, their primary role in UC and inflammatory bowel disease (IBD) is still unclear (Ligumsky et a[., 1981; Rampton & Hawkey, 1984) for although increased PG production may mediate the inflammation of IBD (Ligumsky et al., 1981), animal and human studies have conversely demonstrated a cytoprotective role for PG in the gut (Hawkey & Rampton, 1985). Furthermore, indomethacin, an inhibitor of PG production, exacerbates rather than ameliorates, active UC (Rampton & Sladen, 1981). We (Satsangi et al., 1986), and others (Rachmilewitz er al., I982), have shown increased peripheral blood mononuclear cell (PBMNC) PGE, production in Crohn’s disease (CD), in which the increased PG production detected in the inflamed mucosa has been attributed to the mononuclear (MN) infiltrates (Zifroni et al., 1983). However, previous work found no such increase in PBMNC PGE, production in UC (Rachmilewitz e f al., 1982), in which polymorphonuclear cells (PMN) predominate at the site of inflammation. Studies in this laboratory have not detected production of PGEz of 6-keto-PGF,, in PMN cultures stimulated with zymosan, lipopolysaccharide, formly-methionyl-leucylphenylalanine, silica or ‘activated’ sera. We have, therefore, studied both PGE, and prostacyclin (PGI,, measured as its hydrolysis product 6-keto-PGE,,) in 24 h PBMNC cultures from 22 patients with UC and 14 healthy subjects (HS). Ethical approval was obtained and all patients gave informed consent. Blood cells were obtained from 40ml of heparinized (preservative-free) venous blood by centrifugation (200g for 10min) and suspended in a 1 : 1 (v/v) mixture of 0.01 MEDTA in phosphate-buffered saline and RPMI- I640 tissue culture medium containing antibiotics. The MN cells were isolated by step gradient centrifugation on Ficoll-Hypaque as described previously (Cason et al., 1986). Monocyte composition (%) of the MN cell preparations was determined by staining for non-specific esterases and viability by Trypan

Blue exclusion. M N cell were resuspended at a density of 1 x 10‘ viable MN/ml of RPMI-1640 medium supplemented with 5 x M-Zmercaptoethanol. Cells, unstimulated or stimulated with either 10 jig of lipopolysaccharide (LPS)/ml or silica, were cultured for 24h at 37°C in an humidified atmosphere of 5 % CO, in air. Culture supernatants were obtained by centrifugation, and stored at -20°C. Supernatants were analysed for PG content by radioimmunoassay using commercially available antisera (PGE, from Sigma Chemical Co. Ltd., Poole, Dorset, U.K.; 6-keto-PGF,, from Seragen Inc., Boston, U.S.A.). Results were analysed by the non-parametric Mann-Whitney U test, and expressed as median and ranges. There was no difference between U C and HS, either in viability (96.6 f 2.65% versus 94.8 f. 5.16%; mean f s.D.), purity (94.4 f 6.56% versus 89.7 k 9.66%) or percentage monocyte composition (9.38 k 3.70 versus 9.01 f 4.72%) of the cell preparations used. The previous finding that M N PGE, production was unchanged in U C was confirmed (Table 1). However, prostacyclin (6-keto-PGF,,) was raised for both non- and LPS-stimulated cultures ( P < 0.02) from UC compared with HS. This increase was not seen in silica-stimulated cultures, suggesting a difference in the mechanism of stimulation of PG production between these two substances. Although the increased M N PG production may reflect leucocyte activation in active disease, this would not explain the differences between C D and UC, nor the selective increase in production of PGI,. These differences may therefore represent an intrinsic defect of M N PG producton that contributes to the inflammation seen in UC. Alternatively the observed altered PG levels may be due to drug therapy, for many (13/22) of the UC patients were receiving sulphasalazine which modifies PG production (Hoult & Page, 1981; Berry & Hoult, 1984; Hillier et al., 1984; Hawkey et al., 1985). Such drug induced increases in PG production may be beneficial in IBD.

Table 1. Mononuclear prostaglandin production in ulcerative procto-colitis Both non-stimulated and LPS stimulated PGI, (measured as 6 KF,,) production were significantly raised in UC patients compared with HS ( P < 0.02, Mann-Whitney U test). Results are medians (range). Prostaglandin production (pmol/106 cells)

Stimulant None

Abbreviations used: UC, ulcerative procto-colitis; PG, prostaglandin; IBD, inflammatory bowel disease. PBMNC. peripheral blood mononuclear cells; CD, Crohn’s disease; MN, mononuclear cells; PMN. polymorphonuclear cells: HS, healthy subjects; LPS, lipopolysaccharide.

Vol. 15

6-keto-PGF,,

PGE,

LPS Silica

HS

uc

HS

uc

1.08 (0.00- 2.01) I .85 (0.72 3.10) 1.95 (0.363.80)

1.22 (0.58-6.58) 2.13 (0.82-1 1.3) I .96 (0.8M.52)

0.30

0.41 (0.161.74) 0.48 (0.28-2.03) 0.43 (0.224.86)

(0.04.44) 0.37 (0.04.72) 0.40 (0.04.66)