Stephen C. L. Koh

Application of porous membrane protected micro-solid-phase-extraction combined with gas chromatography-mass spectrometry for the determination of estrogens in ovarian cyst fluid samples.

A cost effective and environmentally friendly extraction technique using porous membrane protected micro-solid phase extraction (μ-SPE) is described for the extraction of estrogens in cyst fluid samples obtained from cancer patients. A sorbent (ethylsilane (C2) modified silica) (20 mg) was packed in a porous polypropylene envelope (2 cm×1.5 cm) whose edges were heat sealed to secure the contents. The μ-SPE device was conditioned with acetone and placed in a stirred (1:5) diluted cyst fluid sample solution (10 mL) to extract estrogens for 60 min. After extraction, the analytes were desorbed and simultaneously derivatized with a 5:1 mixture of acetone and N,O-bis(trimethylsilyl)-trifluoroacetamide. The extract (2 μL) was analyzed by gas chromatography-mass spectrometry. Various extraction, desorption and derivatization conditions were optimized for μ-SPE. With this simple technique, low limits of detection of between 9 and 22 ng L(-1) and linear range from the detection limits up to 50 μg L(-1) were achieved. The optimized method was used to extract estrogens from cyst fluid samples obtained from patients with malignant and benign ovarian tumors.

Effects on hemostasis after two-year use of low dose combined oral contraceptives with gestodene or levonorgestrel.

We studied 67 healthy women who were randomly allocated to receive third generation gestodene (Gynera®) or second generation levonorgestrel (Microgynon 30®) combina tion of low-dose estrogen oral contraceptives (OCs) for their hemostatic effects over 2 years. Hemostatic changes were ap parent within 3 months of OC use. Hematocrit (Hct) was not affected, but hemoglobin (Hb) concentration decreased by 18 months. Shortened prothrombin time (PT) and activated plasma thromboplastin time (APTT) were associated with elevated fi brinogen within the 12-month use of both OCs. Factor VII was reduced only in Micro 30 during the 18 months of use. En hanced thrombin-antithrombin (TAT)-complex level was seen at 18 months of Gynera use. Prothrombin fragment 1+2 (F1+2) rise was seen at 3 months with Micro 30. Reduced antithrombin III (ATIII) activity was seen at 18 months with Gynera and at 24 months with Micro 30. Increased protein C activity was seen at 3 months and reduced protein S occurred at 18 months of Gynera use. Tissue plasminogen activator (t-PA) activity was enhanced for 6 months in both OCs with raised D-dimer levels for 12 months with Gynera and 6 months with Micro 30. De creased t-PA antigen was seen at 18 months and decreased urokinaselike plasminogen activator (u-PA) antigen occurred throughout the 24 months of both OCs use. Enhanced u-PA activity was only seen in Gynera users. Elevated plasminogen levels were apparent throughout both OCs use. PAI-1 levels were significantly decreased with Micro 30. With Gynera, the decreased PAI-1 activity was seen only at 18 months and PAI-1 antigen at 12 months. No change in platelets and von Wil lebrand factor (vWF) were seen in long-term OC use except that β-thromboglobulin (β-TG) showed decreased trends reaching statistical significance by 18 and 24 months of Micro 30 use and by 24 months of Gynera use. A further significant decrease in β-TG, u-PA antigen, ATIII, and protein S levels were seen 3 months after pill stoppage compared with pretreat ment levels. Activated protein C resistance (APCR) was nega tive in all subjects before and during OC use. The study indi cated dynamic balance between coagulation and fibrinolysis with no endothelial activation. However, because some hemo static markers showed wide fluctuations during OC use, a longer term study is warranted to investigate any adverse he mostatic changes that might enhance the risks of venous thromboembolism in Asian subjects known to be less prone to thrombosis. Key Words: Oral contraceptives—Hemostasis.

Coagulation and fibrinolysis in preeclampsia and neonates.

Coagulation and fibrinolysis were determined in 67 Indonesian women admitted to the University Hospitals for delivery in Medan. They were diagnosed to be at term gestation (mean 39.3 ± 1.1 weeks) with moderate and severe preeclampsia (n=32) and in labor, and 8 had preterm labor (gestation mean 33.5 ± 2.6 weeks). Twenty-seven normal pregnant women in labor (gestation mean 39.7 ± 1.0 weeks) served as controls. Cord blood from 23 neonates from normal pregnancy and 31 neonates from preeclampsia was also evaluated. Preeclamptic women in labor showed further enhanced coagulation activation (F1+2) with raised urokinase-like plasminogen activator (u-PA) activity and reduced plasminogen activator inhibitor-2 (PAI-2) levels. In preterm preeclampsia, significantly reduced antithrombin III (ATIII) and PAI-2 levels with further elevated tissue-type PA (t-PA) antigen and plasminogen activator inhibitor-1 (PAI-1) antigen were seen compared to normal pregnancy. These would suggest a state of enhanced thrombin generation with elevated fibrinolytic/inhibitor proteins in preterm preeclampsia. The reduced PAI-2 levels seen in preeclampsia have been suggested to be associated with reduced placental function. Neonates born to mothers of either normal pregnancy or preeclampsia at term showed similar hemostatic changes with reduced fibrinogen, ATIII, t-PA, u-PA antigen, PAI-1 levels, and coagulation activation compared to their respective maternal plasma levels. No significant differences in hemostatic parameters studied between the neonates of both cohorts were seen, and this would suggest that the neonates were protected from the adverse effects of preeclampsia and their hemostatic system was physiologically balanced.

Effects of three types of combined O.C. pills on blood coagulation, fibrinolysis and platelet function

The results of a prospective longitudinal controlled study comparing the coagulation effects of a standard low-dose combined oral contraceptive (Microgynon 30), a desogestrel-containing pill (Marvelon) and a triphasic preparation (Triquilar) after one years treatment in ethnic Chinese women is presented in this paper. In general the changes in coagulation parameters are similar to those seen in Caucasian women. The rise in Factor VII levels seen at 6 and 12 months in Marvelon users was not present to a significant degree in the other pill users. Antithrombin III levels remained unchanged in Microgynon and Marvelon users but with Triquilar there was a significant fall at 6 and 12 months of use. Whether these differences have any clinical relevance in the local Chinese population with low incidence of thromboembolism remains to be seen.

The association between fibrinogen, von Willebrand Factor, antithrombin III, and D-dimer levels and survival outcome by 36 months from ovarian cancer.

Thirty-five patients diagnosed to have ovarian cancer (early FIGO stage I/II n = 11, advanced FIGO stage III/IV, n = 24) were evaluated for hemostatic parameters relating to survival outcome by 36 months of disease. Systemic plasminogen activators and inhibitors were evaluated and we found no significant association with survival outcome and eventually only fibrinogen, von Willebrand Factor (vWF), antithrombin III (ATIII), and D-dimer levels were determined for their association with disease outcome by 12 months, 24 months, and 36 months. Twenty-four patients succumbed to the disease by 36 months (early n = 2, advanced n = 22). The 11 surviving patients (advanced n = 3, including one deceased at 52 months) is still living past 36 months and 82 months at the time of analysis. Elevated fibrinogen, vWF, and D-dimer together with reduced ATIII levels were found to be associated with poor survival outcome by 12 months of disease. Moreover, elevated vWF and D-dimer with reduced ATIII levels was strongly implicated with poor survival outcome by 36 months from ovarian cancer. The overall survival rate at 36 months from ovarian cancer was 31.4%. It is therefore suggested that fibrinogen, vWF, ATIII, and D-dimer levels be used together as prognostic markers for disease outcome especially in patients with advanced ovarian cancer within 36 months of disease. An expanded study is required to confirm these findings.

Plasminogen Activators, Plasminogen Activator Inhibitors and Markers of Intravascular Coagulation in Pre-Eclampsia

In pre-eclampsia (PE), reduced levels of plasma urokinase-like plasminogen activator (u-PA) and plasminogen activator inhibitor-2 (PAI-2), and increased levels of plasma tissue-type plasminogen activator (t-PA) antigen were seen. The majority of moderate and severe pre-eclamptic women (7 out of 10) ended up with pre-term delivery as compared with 2 out of 11 who went on to term. Patients with moderate and severe PE had significantly lower levels (mean +/- SD, ng/ml) of PAI-2 (58.4 +/- 34.9) and u-PA antigen (1.61 +/- 0.62) as compared to those with mild PE (95.6 +/- 39.3 and 1.61 +/- 0.62 and 2.12 +/- 0.61, respectively). Significantly raised t-PA antigen (14.6 +/- 5.7 ng/ml) was seen in moderate and severe PE as compared with mild PE (9.9 +/- 3.4 ng/ml). PAI-1 activity was significantly raised only in moderate and severe PE as compared with normal pregnancy. There were no significant differences in thrombin-antithrombin-III complexes, D-dimer and beta-thromboglobulin levels between the PE group and normal pregnancy, although these parameters were above the non-pregnant levels. Platelets in PE were within the range found in normal pregnancy. It appears that measurements of plasma u-PA and PAI-2 levels in patients with PE may have prognostic value in determining the outcome of pregnancy in this pregnancy disorder.

Two-year follow-up of changes in clinical chemistry in Singaporean Norplant-2 rod acceptors: haemostatic changes.

In this longitudinal study involving 100 Singaporean acceptors, the effects of Norplant on metabolic function was evaluated. With respect to liver function, the results at the end of two years still indicate the presence of possible hepatocellular dysfunction. Despite a 4.5% decrease in serum bilirubin during the second year, the mean serum bilirubin still remained significantly raised (p less than 0.001). Furthermore there was a significant fall in total proteins and globulin during the period under review (p less than 0.001). However mean levels of all three parameters remained within the normal clinical range. As regards lipid metabolism, the total triglycerides and total cholesterol still remained significantly decreased at the end of two years (p less than 0.001). The LDL-cholesterol, which was significantly decreased at the end of one year, increased by 2.2% during the second year. However, this rise in LDL-cholesterol was not significant and was still below the preinsertion level. The HDL-cholesterol levels returned to their preinsertion levels after a significant increase in the first year. The HDL-cholesterol/Total cholesterol - HDL-cholesterol ratio returned to its preinsertion value at the end of two years while the LDL-cholesterol/HDL-cholesterol ratio was still significantly lower than the preinsertion ratio (p less than 0.05). As we have not incorporated a simultaneous non-steroid treated control group, the observed changes in lipoprotein lipids could be attributed to extraneous factors such as diet, exercise or other environmental changes.(ABSTRACT TRUNCATED AT 250 WORDS)

A plasmin generation method for the determination of tissue plasminogen activator (t-PA) activity in blood.

A plasmin generation method to determine tissue plasminogen activator (t‐PA) activity in plasma is described, A protein solution of homogenized fibrin was used as a stimulator in the presence of plasminogen and the plasmin generated was measured by the release of para‐Nitroanilide (p‐NA) from the chromogenic substrate S‐2251. Plasmin generation by 5 iu/mL t‐PA in the presence of 1 CU/mL of plasminogen and 850μg/mL of fibrin solution reaches a peak at about 5 h incubation whilst in plasma, plasmin generation peaks after about 16 h incubation. The highest t‐PA activity in plasma was determined using an assay involving 18 h incubation. In the 21 subjects studied by this method the t‐PA activity at rest ranged from 0·34 to 0·92 iu/mL, with a mean of 0·57 ± 0·15 iu/mL of plasma whilst in the post‐occlusion state the activity ranged from 1·12 to 18·0 iu/mL, with a mean of 5·25 ± 4·49 iu/mL of plasma. We also found that subjects who developed petechiae during occlusion had significantly higher t‐PA activity both at pre‐ and post‐occlusion when compared with those who did not develop petechiae. The t‐PA activity of acid‐treated plasma stored at — 70°C showed no significant changes in activity after 12 weeks of storage when compared with the t‐PA activity of the same plasma tested prior to storage.

Preeclampsia: Haemostatic Status and the Short‐Term Effects of Methyldopa and Isradipine Therapy

Objective: To determine the haemostatic status in preeclampsia and to investigate the effects of short‐term use of anti‐hypertensive drugs, methyldopa and isradipine.

Hemostatic status and fibrinolytic response potential at different phases of the menstrual cycle

Coagulation and fibrinolytic variables including platelet function and endogenous fibrinolytic response were determined in 30 normal healthy women volunteers not on any known medication during the period of study. They were between 18 years and 38 years old and had normal menstrual cycles of between 28 days and 30 days. Blood samples were obtained within one menstrual cycle and after having fasted overnight within days 1 to 3 (menstruation), 5 to 9 (follicular), 10 to 14 (mid-cycle), and 21 to 26 (luteal) of the menstrual cycle. Analysis of variance (ANOVA) showed no significant differences in the hemostatic parameters studied between the phases of the menstrual cycle except for a reduced D-dimer level at mid-cycle. Significant fibrinolytic response was seen after venous occlusion but they were not significantly different between the phases of the menstrual cycle. The women were then divided into either normal weight (n=22) or overweight (n=8) according to World Health Organization (WHO) classification and the data reanalyzed. Elevated tissue plasminogen activator antigen and plasminogen activator inhibitor-1 levels except at menstruation and total protein S except at follicular phase were observed in overweight women together with increased plasminogen level only at luteal phase. Significant endogenous fibrinolytic response seen during the menstrual cycle was not different between normal and overweight women. The study demonstrated that systemic coagulation, fibrinolysis, and platelet function were probably not influenced by natural hormonal changes occurring during the menstrual cycle except for an associated reduced fibrinolytic state at mid-cycle. The hemostatic system in this small group of healthy overweight women studied appeared to be physiologically compromised.