Acute hyperhomocysteinaemia and endothelial dysfunction

RESEARCH LETTERS

Longitudinal hysteroscopic follow-up during tamoxifen treatment P Neven, X De Muylder, Y Van Belle, I Van Hooff, G Vanderick

Tamoxifen is the first choice adjuvant treatment for postmenopausal women with breast cancer. Experimental,1 animal,2 double-blind randomised trials, and other clinical studies3 show that long-term tamoxifen intake can lead to endometrial growth and is a risk factor for the development of endometrial polyps and endometrial cancer. We have previously reported the only known case of new development of an endometrial cancer in a woman who was regularly followed up while on tamoxifen therapy: hysteroscopy and endometrial biopsy initially showed no detectable uterine lesions in 16 women, but after 36 months on tamoxifen, one woman developed a high-grade endometrial cancer. 4 However, undetectable malignant cells could have been present at the time of her base-line assessment. We, therefore, tested 57 postmenopausal women with breast cancer in a similar way at our clinic (table). All the women had tamoxifen prescribed at a daily dosage of 20 mg and had an atrophic endometrium and empty uterine cavity. They were regularly assessed by means of outpatient panoramic hysteroscopy with carbon dioxide as the distention medium. In the event of any woman developing endometrial stimulation or an intrauterine lesion in any of the subsequent hysteroscopies, we carried out an endometrial biopsy. Endometrial polyps were removed and in some cases a hysterectomy was indicated. For the statistical analysis, patients were divided into three groups according to duration of tamoxifen use: less than 3 years; 3–6 years; and longer than 6 years. We used the ␹2 test. We took as significant a p value of less than 0·05. The initial atrophic endometrium showed stimulation in 33 women, whereas the uterine cavity remained atrophic in 24 women on long term tamoxifen intake. There were three endometrial cancers, all in the women on tamoxifen for longer than 6 years. One was a polyp cancer surrounded by a glandulocystic atrophic mucosa, one was a low-grade, and one a high-grade endometrial cancer. Two women developed endometrial hyperplasia and in 28 women the mucosa was “tamoxifen-like”—ie, a smooth white but hypervascularised atrophic endometrial layer with scattered protuberances (an atrophic epithelium overlying stromal oedema, also called glandulocystic atrophia). 20 women developed a benign endometrial polyp mostly of the glandulocystic type; in five the surrounding mucosa was atrophic, in 13 it was tamoxifen-like, and two had patches of endometrial hyperplasia. In this uncontrolled cohort study, there is no comparator cohort of non-tamoxifen-exposed breast-cancer patients to differentiate background events that may be unrelated to tamoxifen. Women who are symptom-free harbour undetected silent endometrial cancers,5 which limits the validity of the results with regard to a cause-effect relation of tamoxifen in all cancer events in this study. The effect of time, Time on tamoxifen (years)

Total

<3 (n=17)

3–6 (n=26)

>6 (n=14)

Endometrium Atrophic Glandulocystic atrophia Hyperplasia (cancer*)

7 10 0

11 14 1

6 4 4 (3)

24 28 5

Uterine cavity Empty Polyp (cancer*)

12 5

16 10

6 8 (3)

34 23

*Number of cancers in parentheses. ␹2<6 years vs >6 years for uterine cavity p=0·14, relative risk 1·64 (95% CI 0·89–3·02).

Endometrial lesions after tamoxifen therapy starting with an empty and atrophic uterine cavity

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as defined by the three different time-periods, to develop endometrial stimulation or endometrial lesions such as polyps or cancer was not significant. However, comparison of women on tamoxifen for up to 6 years versus longer than 6 years, showed a trend towards developing intrauterine lesions, but this association was not significant (p=0·14). Eight out of the 14 women with an initial atrophic endometrium who used tamoxifen for longer than 6 years developed an endometrial lesion, three of which were endometrial cancers. During the 3 years after a normal baseline endometrium, postmenopausal women on tamoxifen 20 mg daily did not develop endometrial hyperplasia or endometrial cancer; however, in this time, benign endometrial polyps and glandulocystic endometria may occur. We thank René Tonglet (Epidemiology Unit, University Clinic, Louvain, Belgium) for statistical advice. 1

2

3

4

5

Anzai Y, Holinka CF, Kuramoto H, Gurpide E. Stimulatory effects of 4-hydroxytamoxifen on proliferation of human endometrial adenocarcinoma cells. Cancer Res 1989; 49: 2362–65. Satyaswaroop PG, Zaino RJ, Mortel R. Estrogen-like effects of tamoxifen on human endometrial carcinoma transplanted into nude mice. Cancer Res 1984; 44: 4006–10. Assikis VJ, Neven P, Jordan VC, Vergote I. A realistic clinical perspective of tamoxifen and endometrial carcinogenesis. Eur J Cancer 1996; 32A: 1464–76. Neven P, De Muylder X, Van Belle Y, Vanderick G, De Muylder E. Hysteroscopic follow-up during tamoxifen treatment. Eur J Obstet Gynaecol Rep Biol 1990; 35: 235–38. Horwitz RI, Feinstein AR, Horwitz SM, Robboy SJ. Necropsy diagnosis of endometrial cancer and detection-bias in case/control studies. Lancet 1981; ii 66–68.

Algemene Kliniek St.-Jan, Departments of Obstetrics and Gynaecology (P Neven) and Histopathology, B-1000 Brussels, Belgium

Acute hyperhomocysteinaemia and endothelial dysfunction John C Chambers, Andrew McGregor, Jeff Jean-Marie, Jaspal S Kooner

Hyperhomocysteinaemia is a major and independent risk factor for vascular disease,1,2 and venous thrombosis. High concentrations of homocysteine are found in up to 30% of patients with atherosclerosis, and concentrations only 12% above the upper limit of normal (15 µmol/L, mild hyperhomocysteinaemia) are associated with a three-fold increase in the risk of acute myocardial infarction. Homocysteine concentrations are determined by genetic and nutritional factors; mutations in the genes for enzymes involved in homocysteine metabolism and deficiencies of vitamins B6, B12, and folic acid are associated with hyperhomocysteinaemia. The mechanisms by which hyperhomocysteinaemia promote atherosclerosis are not fully understood. We have measured flow-mediated dilatation during an oral methionine load to test whether an acute increase in homocysteine concentration is associated with endothelial dysfunction in healthy volunteers. We recruited 13 healthy volunteers (median age 34 [range 21–59] years) with normal blood pressure, cholesterol, glucose, red-cell folate, and vitamin B12 concentrations; none were taking medications. We measured brachial-arterydiameter responses to hyperaemic flow (endothelium dependent), and glyceryltrinitrate (endothelium independent), at 0 h (fasting), 2 h, and 4 h after oral methionine (Lmethionine 100 mg/kg in fruit juice), and, on a separate occasion, after placebo (methionine-free fruit juice). Brachial artery dilatation was measured with a 7·0 MHz linear array transducer, an Acuson 128XP/10 system (Mountain View, California, USA), and a high-resolution ultrasonic vessel-wall tracking system (Vadirec, Ingenious Systems, Netherlands) as

THE LANCET • Vol 351 • January 3, 1998

RESEARCH LETTERS 4

10 8 Flow-mediated dilatation (%)

5

6

National Heart and Lung Institute, Imperial College School of Medicine, Hammersmith Hospital, London W12 0NN, UK (J S Kooner); and Department of Cardiology, Ealing Hospital, Middlesex

4 2 0

Analysis of the ␣-synuclein G209A mutation in familial Parkinson’s disease

–2 –4 –6 –8 0

10 20 30 40 50 Total plasma homocysteine (µmol/L) Flow-mediated dilatation and homocysteine concentration for each of 13 volunteers

described by Celermajer and colleagues.3 We collected blood at 0, 2, and 4 h during the study to measure total plasma homocysteine by high-performance liquid chromatography. Mean flow-mediated dilatation fell after methionine (4·6 [SE 0·8]%, at baseline to 0·7 [1·4]% and –1·3 [0·8]%, at 2 h and 4 h, respectively) but not after placebo (5·2 [1·4]% at baseline, 6·6 [1·5]% and 4·8 [1·3]%, at 2 h and 4 h, respectively; p<0·001). By contrast, there was no significant difference in glyceryltrinitrate-induced brachial-artery dilatation after methionine (22·4 [1·7]%, 19·8 [1·6]% at baseline and 4 h, respectively) or placebo (20·9 [1·6]% and 20·8 [1·8]% at baseline and 4 h, respectively). There was a linear increase in mean plasma homocysteine concentration after oral methionine (from 9·3 [1·0]% at baseline to 24·1 [3·6] and 30·5 [3·0] ␮mol/L at 2 and 4 h, respectively; p<0·0001). Flow-mediated dilatation was strongly related to plasma homocysteine (p<0·001, figure), with no independent effect of time. Our main findings are that an acute increase in plasma homocysteine is associated with substantial impairment of endothelial function in healthy human volunteers and that this relation is inverse and linear. Brachial artery flow-mediated dilatation is endothelium dependent and largely mediated by nitric oxide.4 Our findings, therefore, suggest impaired endothelial nitric-oxide activity in healthy individuals during acute hyperhomocysteinaemia. Endothelial dysfunction occurred at concentrations of homocysteine that were only two-fold higher than the fasting state, and similar to those associated with an increased risk of acute myocardial infarction, stroke, and venous thrombosis. These findings may help to explain the incremental risk of vascular events with increasing homocysteine concentrations,1,2 and accord with previous reports of dose-dependent and time-dependent effects of homocysteine on endothelial cellular function. Invitro studies show that exposure of endothelial cells to homocysteine results in oxidative effects, including generation of superoxide anion radicals and hydrogen peroxide,5 which lead to inactivation of nitric oxide and endothelial-cell damage. The resultant endothelial dysfunction may then contribute to vasospasm, thrombosis, and progression of atherosclerosis. 1

2

3

Joannides R, Haefeli WE, Linder L, et al. Nitric oxide is responsible for flow-dependent dilatation of human peripheral conduit arteries in vivo. Circulation 1995; 91: 1314–19. Loscalzo J. The oxidant stress of hyperhomocyst(e)inemia. J Clin Invest 1996; 98: 5–7.

Clarke R, Daly L, Robinson K, et al. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med 1991; 324: 1149–55. Nygard O, Nordrehaug JE, Refsum H, Ueland PM, Farstad M, Vollset SE. Plasma homocysteine levels and mortality in patients with coronary artery disease. N Engl J Med 1997; 337: 230–36. Celermajer DS, Adams MR, Clarkson P, et al. Passive smoking and impaired endothelium-dependent arterial dilatation in healthy young adults. N Engl J Med 1996; 334: 150–45.

THE LANCET • Vol 351 • January 3, 1998

Sepideh Zareparsi, Jeffrey Kay, Richard Camicioli, Patricia Kramer, John Nutt, Thomas Bird, Michael Litt, Haydeh Payami

Parkinson’s disease (PD) is a movement disorder that affects 1–2% of the population over the age of 60. Recently, Polymeropoulos et al identified a mutation in the ␣-synuclein gene on chromosome 4q21-23, that segregated with PD in a large Italian (Contursi) kindred, and three Greek kindreds.1 The mutation is a base-pair change from G to A at position 209 (G209A). Polymeropoulos et al suggest that this mutation may account for a substantial proportion of early-onset familial PD, 1 in which case a simple mutation screen may prove to be a highly reliable clinical tool for diagnosis of PD and for presymptomatic risk assessment. We screened 65 affected members from 40 white PD kindreds, of northern European origin, for the G209A mutation. The mean onset of PD was 51·4 (SD 17·5) years in the 65 cases. In 14 of the 40 kindreds, the mean onset age was less than 50 years. 19 of the 40 kindreds had three to 15 affected individuals, and the mean onset age was 57·9 (15·7) years. In seven kindreds, one or more members had onset less than 46 years, which was the mean in the Contursi kindred. In addition, we studied 21 smaller families with only two affected individuals, because onset was less than 46 years in at least one relative. The kindreds were ascertained and evaluated at Oregon Health Sciences University and the University of Washington, after giving informed consent. All 40 probands and seven relatives were diagnosed by us using the Hughes et al criteria with one exception: family history was not taken as an exclusion criteria.2 The remaining 18 relatives were diagnosed by community physicians. Necropsy confirmation was available on the two subjects who have died, and in both, Lewy bodies were present in the substantia nigra. Age at onset was defined as the age at which the patient noticed the first PD symptom. The mutation analysis was carried out as described in Polymeropoulos et al.1 We included PUC 19 DNA in all reactions, to ensure that lack of evidence for the mutation was not due to incomplete digestion. None of the subjects had the G209A mutation. The absence of this mutation in these 40 kindreds argues against it being a common cause of PD in families of northern European origin. Also, it implies that this mutation may not be of widespread use for diagnostics in clinical practice. This finding was not surprising because the Contursi kindred is highly unusual, having 60 affected members, high penetrance (~90%), and a rapid course from onset to death despite levodopa responsiveness. The presence of the mutation in three Greek kindreds suggests that this mutation may be more prevalent in PD families of Mediterranean and southern European origin. To determine if PD in our kindreds is caused by another mutation in the ␣-synuclein gene, we carried out linkage analysis in the nine largest kindreds, with the same model

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