Robert N. Fontaine

Metformin-induced resumption of normal menses in 39 of 43 (91%) previously amenorrheic women with the polycystic ovary syndrome

In 43 amenorrheic women with polycystic ovary syndrome (PCOS), 31 (74%) with fasting hyperinsulinemia (> or =20 microU/mL), our aim was to determine whether Metformin (Bristol-Myers Squibb, Princeton, NJ), which reduces hyperinsulinemia, would reverse the endocrinopathy of PCOS, allowing resumption of regular normal menses. A second aim was to assess the effects of weight loss versus other Metformin-induced effects on ovarian function, and to determine if there were different responses to Metformin between those who lost weight and those who did not. A third aim was to assess associations between PCOS, 4G/5G polymorphism in the promoter sequence of the plasminogen activator inhibitor-1 gene (PAI-1 gene), and PAI activity (PAI-Fx). Of the 43 women, 40 (93%) had normal fasting blood glucose and 37 had normal hemoglobin A1C (HgA1C); onlythree (7%) had type 2 diabetes mellitus. Metformin (1.5 to 2.25 g/d) was given for 6.1+/-5.1 months (range, 1.5 to 24), to 16 patients for less than 3 months, to 12 for 3 to 6 months, and to 15 for at least 6 months. On Metformin, 39 of 43 patients (91%) resumed normal menses. The percentage of women resuming normal menses did not differ among treatment duration groups (P<.1) or among dose groups (P>.1). The body mass index (BMI) decreased from 36.4 + 7 Kg/m2 at study entry to 35.1+/-6.7 on Metformin (P=.0008). Of 43 patients, 28 (67%) lost weight (1 to 69 pounds), with nine (21%) losing at least 12 pounds. On Metformin, the median fasting serum insulin decreased from 26 microU/mL to 22 (P=.019), testosterone decreased from 61 ng/dL to 47 (P=.003), and estradiol increased from 41 pg/mL to 71 (P=.0001). Metformin-induced improvements in ovarian function were independent of weight loss (testosterone decrease, P<.002; estradiol increase, P<.0004). The change in response variables on Metformin did not differ (P>.05) between those who lost weight and those who did not, excepting Lp(a), which increased 4 mg/dL in those who lost weight and decreased 9 mg/dL in those who did not (P = .003). The change in response variables on Metformin did not differ among the five quintiles of weight loss, excepting fasting glucose (P<.05), which increased 6 mg/dL in those who lost the least weight on Metformin versus those in the 60th to 80th percentile for weight loss, in whom glucose decreased 33 mg/dL. Although the pretreatment fasting serum insulin was not significantly correlated with testosterone (r=.24, P=.13) or androstenedione (r=.27, P=.09), on Metformin, the change in insulin correlated positively with the change in testosterone (r=.35, P=.047) and with the change in androstenedione (r=.48, P=.01). Patients were more likely than normal controls (83% v 64%, P=.016) to be heterozygous or homozygous for 4G polymorphism of the PAI-1 gene and were also more likely to have high PAI-Fx (> or =22 U/mL, 28% v3%, chi(2)=10.1, P=.001). Metformin reduces the endocrinopathy of PCOS, allowing resumption of normal menses in most (91%) previously amenorrheic women with PCOS.

Hypofibrinolysis, thrombophilia, osteonecrosis.

In the context of additional characterization of the pathoetiologic associations of heritable hypofibrinolysis and thrombophilia with osteonecrosis of the hip, the authors assessed 15 women and 21 men at entry to a 12-week treatment study of the amelioration of Ficat Stages I or II osteonecrosis by low molecular weight heparin (Enoxaparin). All 36 patients had osteonecrosis of the hip; four patients had unifocal osteonecrosis, 25 patients had two joints affected, five had three affected joints, and two had four affected joints. In 11 of 15 women (73%), hyperestrogenemia of pregnancy (20%) or exogenous estrogen supplementation (53%) were associated with the development of osteonecrosis. Five gene mutations affecting coagulation and nine serologic coagulation tests were studied. Compared with control subjects, patients were more likely to have heterozygosity and homozygosity for the hypofibrinolytic 4G polymorphism of the plasminogen activator inhibitor-1 gene. Moreover, the plasminogen activator inhibitor-1 gene product, plasminogen activator inhibitor activity, the major determinant of hypofibrinolysis, was 10 times more likely to be high (> 21.1 U/mL) in patients than in control subjects (31% versus 3%), with a median of 15.7 versus 6.3 U/mL. Compared with controls, patients were more likely to have the thrombophilic methylenetetrahydrofolate reductase gene mutation. In addition, the thrombophilic methylenetetrahydrofolate reductase gene product, homocysteine, was four times more likely to be high (> 13.5 umol/L) in patients than in control subjects (20% versus 5%), with a median of 9.1 versus 7 umol/L. Twenty-three percent of patients had low levels (< 65%) of the thrombophilic free protein S versus 3% of control subjects. Patients were more likely than control subjects to have hypofibrinolytic high lipoprotein (a) (≥ 35 mg/dL), 33% versus 13%. Median lipoprotein (a) was higher in patients than in control subjects, 15 versus 5 mg/dL. Heritable hypofibrinolysis and thrombophilia, often augmented in women by hyperestrogenemia, seem to be major pathoetiologies of osteonecrosis. If the association between coagulation disorders and osteonecrosis reflects cause and effect, as postulated, then anticoagulation with Enoxaparin should be a promising therapy for patients with osteonecrosis.

Metformin to restore normal menses in oligo-amenorrheic teenage girls with polycystic ovary syndrome (PCOS)

PURPOSE To describe our clinical experience in using Metformin combined with a high protein-low carbohydrate diet to restore normal menstrual cycles in teenaged females with polycystic ovary syndrome (PCOS). METHODS To enter the study, patients had to have well-documented PCOS, be oligo- (six cycles or less in the preceding year) or amenorrheic (absence of menstrual cycles for 1 year), and not have exclusionary diseases or drugs. Accompanying a high protein-low carbohydrate diet, Metformin (1.5-2.55 g/day) was given for 10.5 +/- 6.4 months (range, 4.5-26.5 months). Follow-up every 8-10 weeks for > or =6 months was scheduled with interval history, review of menstrual status, assessment of any Metformin-related side effects, brief physical examination, and determination of weight and blood pressure. RESULTS All 11 girls had normal fasting blood glucose and glycohemoglobin. Pre-Metformin, five girls were amenorrheic, three had only one menstrual cycle in the previous year, and three had > or =6 cycles/year. With Metformin, 10 of 11 girls (91%) resumed regular normal menses; 39% of 38 follow-up visits with regular normal menstrual cycles were ovulatory with normal luteal-phase serum progesterone (> or =2.3 ng/mL). Of the 11 girls, nine (82%) lost weight; five girls lost > or =11 lb and seven lost > or =5 lb. After adjusting for weight reduction, with Metformin, estradiol and progesterone rose (p =.0014,.027, respectively) (changes consistent with resumption of regular normal menses), total plasma cholesterol fell (p =.026), and there was a downward trend in testosterone (p =.068). CONCLUSION Metformin safely ameliorates the endocrinopathy of PCOS in previously oligo-amenorrheic teenage females with PCOS, facilitating resumption of normal menses in most girls.

Effect of Exogenous Estrogen on Atherothrombotic Vascular Disease Risk Related to the Presence or Absence of the Factor V Leiden Mutation (Resistance to Activated Protein C)

Estrogen replacement therapy (ERT), which produces acquired resistance to activated protein C when superimposed on heritable resistance to activated protein C (the mutant Factor V Leiden trait), may promote venous and arterial thrombosis. In a cross-sectional study of 423 women referred for hyperlipidemic therapy (93 of whom [22%] were on ERT), our specific aim was to determine whether ERT and heterozygosity for the Factor V Leiden mutation and/or resistance to activated protein C interacted as risk factors for atherothrombosis. Of the 423 women, 168 (40%) had atherothrombosis, 19 (4%) were heterozygous for Factor V Leiden mutation or had resistance to activated protein C <2 (Factor V Leiden mutation+), and 404 were wild-type normal for the Factor V gene and/or had resistance to activated protein C > or =2 (Factor V Leiden mutation-). By stepwise logistic regression, positive explanatory variables for atherothrombosis included hypertension (p = 0.002), age (p = 0.003), relatives with atherothrombosis (p = 0.002), anticardiolipin antibody immunoglobulin-M (p = 0.02), and a Factor V Leiden mutation*ERT interaction term where atherothrombosis events were more likely in 2 subgroups of women (ERT- and Factor V Leiden mutation-) or (ERT+ and Factor V Leiden mutation+) (p = 0.02). High-density lipoprotein cholesterol was inversely associated with atherothrombosis (p = 0.004). In a separate logistic regression model for the 213 women with a polymerase chain reaction measurement of the Factor V gene, ERT was protective (p = 0.008); the Factor V Leiden mutation was positively associated with atherothrombosis (p = 0.05). The atherothrombosis odds ratio risk for ERT (yes vs no) was 0.36 (95% confidence intervals [CI] 0.16 to 0.74, p = 0.007). The atherothrombosis risk odds ratio in women heterozygous for the Factor V Leiden mutation (vs normal) was 2.00 (95% CI 1.02 to 4.22, p = 0.05). ERT may be protective against atherothrombosis when the Factor V Leiden mutation is absent, whereas the Factor V Leiden mutation may increase risk for atherothrombosis, particularly in the presence of ERT. We suggest that the Factor V Leiden mutation be measured in all women on ERT or before beginning ERT to identify those heterozygous for the Factor V Leiden mutation (4%), in whom ERT is relatively or absolutely contraindicated because of increased risk for atherothrombosis and thromboembolism. A second, much larger group of women will also be identified without the factor V Leiden mutation (96%), in whom ERT may reduce the risk for atherothrombosis.

Association between the T-786C eNOS polymorphism and idiopathic osteonecrosis of the head of the femur.

BACKGROUND Nitric oxide regulates bone turnover by osteoblasts and osteoclasts. Nitric oxide production is impaired by the T-786C eNOS single nucleotide polymorphism, with a substitution of the nucleotide thymine by cytosine at a locus 786 base pairs upstream of the eNOS gene. This leads to vasoconstriction, platelet aggregation, reduced angiogenesis, and reduced bone formation, all of which may be associated with osteonecrosis of the hip. We studied relationships between the T-786C eNOS polymorphism and idiopathic and secondary necrosis of the head of the femur in order to better understand the pathophysiology of osteonecrosis. METHODS With use of polymerase chain reaction methodology, the T-786C eNOS polymorphism was compared in ninety-five patients with femoral head necrosis (including thirty-six nonsmokers with idiopathic necrosis and fifty-nine patients with secondary necrosis) and seventy-two healthy normal adult controls. RESULTS Homozygosity for the mutant eNOS allele (TT) was present in eight (22%) of thirty-six patients with idiopathic osteonecrosis as compared with one (3%) of thirty-six race, gender, and age-matched controls; heterozygosity (TC) was present in nineteen patients (53%) as compared with ten controls (28%); and the wild-type normal genotype (CC) was present in nine patients (25%) as compared with twenty-five controls (69%) (p = 0.0004). Logistic regression revealed that the T-786C eNOS mutant allele was positively associated with idiopathic osteonecrosis of the femoral head (odds ratio, 6.0; 95% confidence interval, 2.51 to 14.4). The fifty-nine patients with secondary osteonecrosis did not differ from fifty-two race, gender, and age-matched controls in terms of the distribution of the T-786C eNOS polymorphism (p = 0.19) or in terms of mutant allele frequency (30% compared with 21%; p = 0.15). The thirty-six patients with idiopathic osteonecrosis differed from the fifty-nine patients with secondary osteonecrosis in that they were more likely to have mutant eNOS genotypes (p = 0.033) and to have a higher mutant T allele frequency (49% compared with 30%; p = 0.009). CONCLUSIONS The T-786C eNOS polymorphism and resultant reduction of nitric oxide production is associated with, and may contribute to, the pathogenesis of idiopathic osteonecrosis of the femoral head. LEVEL OF EVIDENCE Prognostic Level III. See Instructions to Authors for a complete description of levels of evidence.

Prospective 10-year evaluation of hypobetalipoproteinemia in a cohort of 772 firefighters and cross-sectional evaluation of hypocholesterolemia in 1,479 men in the National Health and Nutrition Examination Survey I

Our specific aim in a 10-year prospective study of 772 Cincinnati firemen (predominantly aged 26 to 46 years) was to determine the prevalence, attributes, and etiology of persistent hypobetalipoproteinemia, defined by entry low-density lipoprotein cholesterol (LDLC) less than 75 mg/dL. A second specific aim was to cross-sectionally assess hypocholesterolemia (defined by total serum cholesterol [TC] < 130 mg/dL) in 1,314 white and 165 black men aged 26 to 46 years in the National Health and Nutrition Examination Survey (NHANES I). The 141 black and 631 white firemen had 4,973 person-years of follow-up time (median, 7.1 yr/man). Of 772 men, 44 (5.7%) had entry LDL levels less than 75 mg/dL; they had a mean follow-up time of 7.3 yr/man. Of these 44 men, there were 12 (1.8% of the cohort) with entry LDLC less than 75 mg/dL, and at least 67% of their follow-up LDLC levels were less than 75. Their mean entry TC and LDLC levels were low (130 and 58 mg/dL), mean triglyceride (TG) was low (63 mg/dL), and mean high-density lipoprotein cholesterol (HDLC) was high (60 mg/dL), LDLC remained at less than 75 mg/dL in 81% of their follow-up samples. Their mean entry and follow-up cholesterol and LDLC did not differ (P > .1, 130 v 133 mg/dL and 58 v 63 mg/dL). Compared with 32 men with entry LDLC less than 75 mg/dL but with less than 87% of follow-up LDLC less than 75 mg/dL, the 12 men with persistently low LDLC had lower mean Quetelet indices and diastolic blood pressure at entry (2.36 v 2.58, P = .056; 73 v 80 mm Hg, P = .03) and on follow-up study (2.45 v 2.69, P = .04; 72 v 79 mm Hg, P = .05). Of 12 men with persistently low LDLC, two had truncated apolipoprotein (apo) B (familial hypobetalipoproteinemia, two had the apo E genotype 2/3, and two had acquired hypobetalipoproteinemia that antedated mortality from melanoma by 9 years and from alcoholism by 2 years. Comparable to white and black firemen aged 26 to 46 years, 2.9% and 3.6% of whom had entry serum TC less than 130 mg/dL, of 1,314 white and 165 black men in the NHANES I study (aged 26 to 46), 1.8% and 3.6% had hypocholesterolemia (entry TC < 130 mg/dL). Daily mean calorie, fat, and protein intake (grams per day) did not differ (P > .05) in men with entry TC less than 130 mg/dL compared with those with TC 130 to 230 or greater than 230 mg/dL. Hypocholesterolemia in white and black men in NHANES I could not be attributed to hypocaloric intake or to protein, fat, or carbohydrate undernutrition. There appear to be racial differences in the prevalence of hypocholesterolemia. Blacks comprised 18% of the firemens cohort but 42% of those with persistent hypobetalipoproteinemia; among NHANES I subjects, 3.6% of blacks were hypocholesterolemic versus 1.8% of whites. Unless persistent hypobetalipoproteinemia reflects an underlying disease, alcoholism, etc., it is often heritable, and may be associated with a reduced likelihood of coronary heart disease (CHD) and with increased longevity.

Anticoagulant therapy for osteonecrosis associated with heritable hypofibrinolysis and thrombophilia.

Osteonecrosis develops as the end-result of reduced blood flow to the femoral head. We postulate that venous thrombosis leads to increased intraosseus venous pressure, reduced arterial flow and hypoxic bone death. Hypofibrinolysis (reduced ability to lyse thrombi) and thrombophilia (increased tendency to form thrombi) appear to play an important role in osteonecrosis. If coagulation disorders cause osteonecrosis, then anticoagulation might ameliorate osteonecrosis. In subjects with coagulation disorders and osteonecrosis of the hip, provided that anticoagulant therapy is started before irreversible segmental collapse of the head of the femur, osteonecrosis may be arrested or, speculatively, sometimes reversed. This has the potential of preventing femoral head collapse which usually leads to total hip replacement.

VKORC1 Variant Genotypes Influence Warfarin Response in Patients Undergoing Total Joint Arthroplasty: A Pilot Study

AbstractWarfarin dosing algorithms do not account for genetic mutations that can affect anticoagulation response. We retrospectively assessed to what extent the VKORC1 variant genotype would alter the likelihood of being a hyperresponder or hyporesponder to warfarin in patients undergoing total joint arthroplasty. We used the international normalized ratio (INR) on the third postoperative day of 3.0 or greater to define warfarin hyperresponders and 1.07 or less to define hyporesponders. A control group of normal responders was identified. From a cohort of 1125 patients receiving warfarin thromboprophylaxis, we identified 30 free of predisposing factors that could affect warfarin response: 10 hyperresponders, eight hyporesponders, and 12 normal responders. Homozygous carriers of the VKORC1 mutant AA genotype were more likely (compared with carriers of GA or GG genotypes) to be hyperresponders (odds ratio, 7.5; 95% confidence interval, 1.04–54.1). Homozygous carriers of the GG (normal) genotype were more likely (compared with carriers of AA or GA genotypes) to be hyporesponders (odds ratio, 9; 95% confidence interval, 1.14–71). Preoperative screening for the VKORC-1 genotype could identify patients with a greater potential for being a hyperresponder or hyporesponder to warfarin. This may allow an adjusted pharmacogenetic-based warfarin dose to optimize anticoagulation, reducing postoperative risks of bleeding and thrombosis or embolism. Level of Evidence: Level III, diagnostic study. See the Guidelines for Authors for a complete description of levels of evidence.

19 STROMELYSIN-1 5A/6A AND eNOS T-786C POLYMORPHISMS, MTHFR C677T AND A1298C MUTATIONS, AND CIGARETTE-CANNABIS SMOKING: A PILOT STUDY OF GENE-ENVIRONMENT PATHOPHYSIOLOGICAL ASSOCIATIONS WITH BUERGER'S DISEASE.

Buergers disease (BD) etiologies are poorly understood. Beyond smoking cessation, medical-surgical treatments have limited success. We hypothesized that mutations associated with arterial vasospasm (stromelysin-1 5A/6A, eNOS T-786C) and C677T-A1298C methylenetetrahydrofolate reductase (MTHFR) interacted with cigarette-cannabis smoking, reducing vasodilatory nitric oxide (NO), promoting arterial spasm-thrombosis. Of 19 smoking BD patients (13 men [2 siblings], ± women, 18 Caucasian, 1 African American), compared to 200 healthy Caucasian controls, BD patients were more likely to have 6A6A stromelysin-1 homozygosity (7/19 [37%] vs 46/200 [23%]) and to have eNOS T-786C homozygosity (3/19 [16%] vs 22/200 [11%]), but these patient-control differences were not significant, p = .4, 0.5. C677T MTHFR homozygosity or compound C677T-A1298C heterozygosity did not differ in patients vs controls (6/19 [32%] vs 70/200 [35%]), p = .8. In 9 patients who stopped and 1 who continued smoking, all stromelysin-1 5A/6A and/or eNOS heterozygotes-homozygotes, lower limb gangrenous ulcers, and intractable ischemic rest pain with arterial occlusion progressed despite conventional medical therapy, threatening amputation. In these 10 patients, to increase vasodilatory NO via endothelial NO synthase, L-arginine (15 g/day) was given, along with folic acid 5 mg, vitamin B6 (100 mg), and B12 (2,000 μg/day) to optimize homocysteine metabolism and reduce asymmetric dimethylarginine, a NO synthase inhibitor. Unexpectedly quickly and strikingly, within 8 weeks to 8 months on L-arginine-folic acid, all 10 treated patients improved with uniform pain reduction, ulcer healing, and in 5, full recovery of previously absent peripheral pulses. In smokers homo-heterozygous for stromelysin-1 5A/6A, eNOS T-786C, and C677T-A1298C MTHFR mutations, we speculate that the development and severity of BD are related to a gene-environment vasospastic interaction with reduced NO-mediated vasodilatation. Increasing NO production by L-arginine while optimizing homocysteine metabolism by folic acid-B6-B12 may have therapeutic benefit. Further blinded, placebo-controlled studies are needed to determine whether our observations can be generalized to larger BD cohorts.

22 STROMELYSIN-1 5A/6A AND eNOS T-786C POLYMORPHISMS, MTHFR C677T-A1298C COMPOUND HETEROZYGOSITY, AND CIGARETTE-CANNABIS SMOKING: GENE-ENVIRONMENT PATHOPHYSIOLOGICAL ASSOCIATIONS WITH BUERGER'S DISEASE

In a 26-year-old white male with Buergers disease we hypothesized that gene polymorphisms which confer susceptibility to arterial spasm (stromelysin-1 5A/6A, eNOS T-786C) and C677T-A1298C MTHFR compound heterozygosity with decreased bioavailability of nitric oxide (NO) interacted with cigarette-cannabis smoking, as gene-environment interactive etiologies for the peripheral artery spasm, thrombosis, and arterial occlusion of Buergers disease. In a 26-year-old male who had heavily smoked cigarettes-cannabis, Buergers disease with rapid progression of bilateral lower limb gangrenous ischemia and intractable ischemic pain on rest despite smoking cessation threatened high above-knee amputations. There was complete arterial occlusion at the level of the left popliteal trifurcation. After documentation of thrombophilic C677T-A1298C compound MTHFR heterozygosity, folic acid 5 mg, vitamin B6 100 mg, and vitamin B12 2000 μg/day were started to optimize homocysteine metabolism and to reduce asymmetric dimethylarginine (ADMA) levels, an endogenous inhibitor of NO synthase. L-Arginine (15 g/day) was given as an approach to increase vasodilatory NO production via endothelial NO synthase (eNOS). Within 8 weeks on therapy, gangrenous ulcers on the right foot and leg healed, pain subsided, and peripheral arterial pulses normalized. Previously existing gangrene of the left great toe and second toe became complicated with cellulitis, necessitating below knee amputation. Healing was subsequently uneventful with bilateral normal peripheral arterial pulses. PCR analyses revealed heterozygosity for stromelysin-1 5A/6A and eNOS T-786C polymorphisms, previously shown to confer susceptibility to coronary artery spasm and myocardial infarction in synergy with cigarette smoking. In cigarette-cannabis smokers, we speculate that the development and severity of Buergers disease are related to a gene-environment interaction in subjects heterozygous for the stromelysin-1 5A/6A and eNOS T-786C polymorphisms, as well as C677T-A1298C MTHFR compound heterozygosity, which altogether reduce endogenous NO-mediated vasodilatation. In such cases, Buergers disease can successfully be treated by increasing NO production by giving oral L-arginine (15 g/day) while also optimizing homocysteine metabolism by provision of folic acid (5 mg), vitamin B6 (100 mg), and vitamin B12 (2000 μg)/day.