Relationship between ovarian cortisol:cortisone ratios and the clinical outcome of in vitro fertilization and embryo transfer (IVF-ET).

OBJECTIVE: Previously, we have reported an association between low levels of intraovarian cortisol metabolism, mediated by 11beta-hydroxysteroid dehydrogenase (11betaHSD), and the establishment of pregnancies by in vitro fertilization and embryo transfer (IVF-ET). The objective of the present study was to investigate the relationship between the clinical outcome of IVF-ET and the intraovarian concentrations of cortisol and cortisone and the cortisol:cortisone ratios in random samples of ovarian follicular fluid (FF). DESIGN: Retrospective, double-blind correlation analyses. PATIENTS: FF samples (n = 41) were obtained from 23 women undergoing gonadotrophin-stimulated IVF-ET cycles at the Cardiff Assisted Reproduction Unit. MEASUREMENTS: Clinical pregnancy was confirmed by ultrasonography. Intrafollicular steroid concentrations were measured by radioimmunoassays. RESULTS: Concentrations of both cortisol and cortisone were significantly lower in FF samples obtained from 6 patients that conceived than in samples obtained from 17 patients that did not achieve pregnancy (cortisol (mean +/- SEM) = 304 +/- 29 vs. 407 +/- 26 nmol/l, P = 0. 0411; cortisone = 32 +/- 3 vs. 65 +/- 7 nmol/l, P = 0.0002). Intrafollicular cortisol:cortisone ratios were significantly higher in samples from conception cycles than in those samples obtained from nonconception cycles (9.7 +/- 0.7 vs. 6.9 +/- 0.5, respectively, P = 0.0060). Whereas 5 of 10 women with intrafollicular cortisol:cortisone ratios greater than the outcome-independent mean of 7.7 became pregnant, only 1 of the 13 patients with intrafollicular cortisol:cortisone ratios < 7.7 conceived (chi2 = 5. 247, P = 0.0220). CONCLUSIONS: Concentrations of both cortisol and cortisone were significantly lower in FF samples obtained from patients that conceived by IVF-ET than in those obtained from nonconception cycles. Conception by gonadotrophin-stimulated IVF-ET was associated with an elevated intrafollicular ratio of cortisol:cortisone, consistent with a low level of intraovarian cortisol oxidation by 11betaHSD.

A randomized controlled trial of weight reduction and exercise for diabetes management in older African-American subjects.

OBJECTIVE: To evaluate a weight loss and exercise program designed to improve diabetes management in older African-Americans. RESEARCH DESIGN AND METHODS: Overweight African-Americans (n = 64) ages 55-79 years with NIDDM were randomized to either an intervention (12 weekly group sessions, 1 individual session, and 6 biweekly group sessions) or usual care (1 individual session, and 6 biweekly group sessions) or usual care (1 class and 2 informational mailings). Clinical and behavioral variables were assessed at 0, 3, and 6 months of treatment. RESULTS: Significant net differences in the intervention versus usual care were observed for weight (-2.0 kg, P = 0.006), physical activity, and dietary intake of fat, saturated fat, cholesterol, and nutrition knowledge at 3 months (all P < 0.05) and for weight at 6 months (-2.4 kg; P = 0.006) and mean HbA1c values at 3 and 6 months (respectively, -1.6 and -2.4%, both P < 0.01). After the adjustment for changes in weight and activity, the intervention participants were approximately twice as likely to have a one unit decrease in HbA1c value as those in usual care. Blood pressure increase sin usual care participants resulted in net differences (intervention minus control) at 3 and 6 months of -3.3 (P = 0.09) and -4.0 (P = 0.05) mmHg diastolic, respectively, and -8.4 (P = 0.06) and -5.9 (P > 0.10) mmHg systolic, respectively. Blood lipid profiles improved more in intervention than usual care participants, but not significantly. CONCLUSIONS: The intervention program was effective in improving glycemic and blood pressure control. The decrease in HbA1c values was generally independent of the relatively modest changes in dietary intake, weight, and activity and may reflect indirect program effects on other aspects of self-care.

Effects of ethanol on tissue folate incorporation and recovery from folate deficiency in rats.

Studies in folate-deficient alcoholics suggest that ethanol interferes with the recovery of folate status and the hematopoietic response to folate. Previous animal studies have suggested diverse effects of ethanol on intestinal absorption, hepatic metabolism, and urinary excretion of folate. In order to examine the effects of ethanol on folate distribution during folate deficiency, tissue incorporation of a tracer dose of folate was studied in rats chronically fed ethanol-containing and/or folate-deficient diets. Rats fed these diets were also used to study the effect of chronic ethanol consumption on the dietary reversal of folate deficiency by changing the diets (adding folate or replacing ethanol) from 12 to 16 weeks. After 16 weeks, tissue folate depletion was severe in rats fed folate-deficient diets. Plasma and whole body retention of the tracer dose of folate was decreased in folate-deficient rats consuming ethanol. In folate-deficient rats, ethanol consumption increased the incorporation of folate by the kidney and brain, but had no effect in other tissues (liver, lung, spleen, intestine, testis). In ethanol-fed folate-deficient rats that continued to consume ethanol, but with added folate in their diets, urine, plasma, liver, and kidney folate levels returned to control levels in 4 weeks. In the rats that stopped ethanol, but continued low folate diet consumption, no recovery of tissue folate levels was seen in 4 weeks. These results suggest that chronic ethanol consumption can exacerbate folate requirements by inhibiting body retention of small doses of folate. However, these effects are minor because ethanol consumption does not block recovery from folate deficiency when rats are fed sufficient amounts of folate.

Incorporation of 3H-label from folic acid is tissue-dependent in folate-deficient rats.

To study the tissue-specificity of folate deficiency, male Sprague-Dawley rats were fed folate-replete or folate-deficient diets with and without sulfonamide for 16 wk, and then injected with [3H]folic acid (1.5 nmol/kg). Rats were killed after 24 h, and the blood, urine and various organs were prepared for analysis of endogenous and 3H-labeled folate. Endogenous folate levels decreased due to folate deficiency to the greatest extent in the urine and plasma, followed by liver, kidney and other tissues (spleen, testis, lung and intestine), but no decrease was noted in the brain. Of all tissues of folate-deficient rats, the brain showed the greatest increase in incorporation of 3H-label from folate relative to folate-replete rats, with the largest effect in rats that were most deficient in plasma folate. Incorporation of label was increased due to folate deficiency in a number of tissues, with an inverse correlation with the tissue folate concentration. In contrast, hepatic [3H]folate incorporation was lower in folate-deficient rats than in folate-replete rats, with a direct correlation between endogenous folate concentration and the incorporation of labeled folate. These results show that the brain and other organs adapt to the development of folate deficiency because of greater incorporation of folate from exogenous sources. The lower incorporation by the liver of folate-deficient rats may result from the greater incorporation by other tissues.

Effects of chronic ethanol and diet treatment on urinary folate excretion and development of folate deficiency in the rat.

Because the folate deficiency of chronic alcoholism has been proposed to result from ethanol-induced effects on metabolism or urinary excretion of folate, the present study was designed to evaluate the role of chronic ethanol-induced urinary folate loss on folate homeostasis in the rat. Male Sprague-Dawley rats were fed nutritionally sufficient liquid diets for 12 wk with or without ethanol, folate and sulfonamide. Urinary folate excretion was increased in ethanol-fed rats consuming folate-containing diets, but not in rats fed folate deficient diets. Consumption of folate-deficient diets led to a rapid decrease in urinary folate excretion, suggesting renal adaptation to conserve folate. Tissue and plasma levels of folate were mostly unaffected by ethanol ingestion in rats fed folate-containing diets. Ethanol treatment did not consistently enhance tissue folate depletion in rats fed folate-deficient diets. The results suggest that in rats consuming diets containing high levels of folate, chronic ethanol ingestion increased urinary folate excretion, but not to a sufficient magnitude to consistently affect folate homeostasis.

Effects of acute ethanol on urinary excretion of 5-methyltetrahydrofolic acid and folate derivatives in the rat.

Acute ethanol treatment enhances the urinary excretion of endogenous folate. This effect has been implicated in the development of folate deficiency associated with chronic alcoholism. Previous studies have shown that urinary excretion of total [3H]-label after administration of [3H]folic acid is slightly higher in ethanol-treated rats because of conversion of the tracer to forms whose excretion is not affected by ethanol. Since [3H]folic acid is not the physiological substrate for the kidney, studies were performed using a high specific activity 5-methyltetrahydrofolic acid ([3H]5-CH3-H4 folic acid). Male Sprague-Dawley rats were given four consecutive hourly doses of ethanol at 1 g/kg, followed by infusion of [3H]5-CH3-H4 folic acid at 5 h. Urine samples were collected to 6 h, when rats were killed, and plasma, liver and kidney samples were collected. Endogenous urinary folate excretion and the fractional urinary excretion of both endogenous and [3H]5-CH3-H4 folic acid at the 5-6 h time period were significantly higher in ethanol-treated rats. The kidney had a tenfold greater specific incorporation of [3H]-label than did the liver. High performance liquid chromatography (HPLC) analysis of the plasma obtained at 6 h showed that 68% of the label was [3H]5-CH3-H4 folic acid, and HPLC analysis of the urine obtained from 5-6 h showed that only 10% of the label was [3H]5-CH3-H4 folic acid. The data indicate that [3H]5-CH3-H4 folic acid was rapidly taken up by the kidney and metabolized to other folate and nonfolate forms, which were then secreted into the renal tubule for excretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Ethylene glycol and glycolate kinetics in rats and dogs.

Ethylene glycol (EG) toxicity results from its metabolism to glycolic acid and other toxic metabolites. The accumulation of glycolate and the elimination kinetics of EG and its metabolites are not well understood, so studies with male Sprague-Dawley rats and mixed breed dogs have been carried out. EG was administered by gavage to rats and dogs, which were placed in metabolic cages for urine and blood sample collection at timed intervals. The peak plasma level of EG occurred at 2 hr after dosing and that of glycolate between 4-6 hr. The rate of EG elimination was somewhat faster in rats with a half-life of 1.7 hr compared to 3.4 hr in dogs. The maximum plasma level of glycolate was greater in rats, although the pattern of accumulation was similar to that in dogs. Glycolate disappeared from the plasma at the same time as EG, suggesting a slower rate of elimination of the metabolite than that of EG. Renal excretion of EG was an important route for its elimination, accounting for 20-30% of the dose. Renal excretion of glycolate represented about 5% of the dose. EG induced an immediate, but short-lived diuresis compared to that in control rats. Minimal clinical effects (mild acidosis with no sedation) were noted at these doses of EG (1-2 g/kg) in both rats and dogs. The results indicate that the toxicokinetics of EG and glycolate were similar in both species.

Differential effects of acute ethanol on urinary excretion of folate derivatives in the rat.

The ethanol-induced increase in the urinary excretion of folate has been determined to be both a time- and dose-dependent phenomenon and it has been speculated that this loss may enhance the development of folate deficiency. However, ethanol has only a minor effect on the renal clearance of exogenously administered [3H]folic acid [3H]PteGlu) in relation to that of inulin. To clarify this variable effect of ethanol, male Sprague-Dawley rats were given four consecutive hourly doses of ethanol at 1 g/kg and placed in metabolic chambers for collection of urine. At 5 hr, [3H]PteGlu was administered and urine samples were collected for 1 hr. At 6 hr, rats were sacrificed with collection of plasma, liver and kidney samples. A significant increase in the urinary excretion of endogenous folates occurred in ethanol-treated rats at both the 4-5- and 5- to 6-hr time periods, but no significant increase in 3H-labeled derivatives was noted in ethanol-treated rats. Subsequent high pressure liquid chromatographic analysis of urine extracts showed that the predominant 3H-labeled PteGlu metabolites were [3H]-5-formimino-H4PteGlu and the formyl-tetrahydrofolates, whereas the major endogenous form was 5-CH3-H4PteGlu. Ethanol administration increased significantly the excretion of the methyl derivative, with minor effects on the other folate forms. These results suggest that there is a selective effect of ethanol on the urinary excretion of specific folate derivatives. Also, inasmuch as no 5-formimino-H4-PteGlu was detected in the plasma, the rodent kidney appears to have the capacity for uptake and metabolism of filtered PteGlu.

Methanol and formate kinetics in late diagnosed methanol intoxication.

In a 21-year-old subject, methanol intoxication was undiagnosed for 12 hours after admission. Only bicarbonate treatment was given during this period, although treatment later included ethanol and haemodialysis. The maximal blood methanol and formate levels were 143 (44.7) and 54.3 mg/dl (11.8 mmol/L), respectively. The delayed diagnosis uniquely allowed for an estimate of methanol elimination kinetics. Before specific treatment, methanol elimination was of zero-order, with a rate of 8.5 mg/dl/h. After admission, the formate levels remained relatively constant until blood pH was normalised by bicarbonate treatment. From this point the formate levels declined, despite an unchanged methanol elimination, indicating that the formate was eliminated faster than it was formed from methanol. Thus, formate elimination may be pH-dependent and aggressive treatment of the acidosis may increase this elimination.

Distribution of oral 4-methylpyrazole in the rat: inhibition of elimination by ethanol.

4-Methylpyrazole (4-MP), a potent competitive inhibitor of alcohol dehydrogenase activity, is being studied as a therapeutic agent for methanol and ethylene glycol poisoning. In order to evaluate the distribution of 4-MP using doses in the potentially therapeutic range, male Sprague-Dawley rats were administered 4-MP orally at zero time in doses of 5, 10, or 20 mg/kg. Half of the rats were also treated orally at 0, 1, 2, and 3 h with ethanol (1 g/kg each h) and half with glucose in isocaloric amounts. At doses of 10 and 20 mg/kg, 4-MP elimination appeared to be saturated, with an elimination rate of 10 mumol/L/h. Elimination at 5 mg/kg was non-conclusive as to the order. The rate of 4-MP elimination was decreased about 50% by concomitant administration of ethanol. Urinary excretion of unchanged 4-MP accounted for only about 1% of the dose; the amount excreted unchanged was significantly increased by ethanol administration. The results demonstrate the mutual inhibition of metabolism by ethanol and 4-methylpyrazole, which may explain why the inhibition of ADH by 4-MP can be longer than that predicted by the elimination rate of 4-MP alone.

Study of dose-dependence and urinary folate excretion produced by ethanol in humans and rats.

Acute ethanol ingestion by human alcoholic subjects produces a marked decrease in serum folate levels within 16 hr. A similar decrease occurs in rats and can be explained by a marked increase in urinary folate excretion following ethanol treatment. To assess the effects of acute ethanol ingestion on urinary folate excretion in healthy human volunteers, two studies were carried out at initial ethanol dose levels of 0.8 g/kg and 1.0 g/kg, respectively. Blood ethanol levels peaked at 70 mg/dl in the first study, but in the second study were 100 +/- 20 mg/dl through 6 hr. Only in the second study were urinary folate levels significantly increased by ethanol administration, and this 8 hr after ingestion. This increase was accompanied by a decrease in urine volume so that in neither study was the total amount of urinary folate excreted from 0-12 hr increased by ethanol ingestion. Studies with various dose levels of ethanol in rats showed that there was a linear dose-response relationship between the total urinary folate excretion and the dose of ethanol. Peak urinary ethanol levels also correlated with urinary folate excretion. These results suggest that doses of ethanol larger than 1.0 g/kg produce increases in urinary folate excretion and that the inability to observe large increases in studies in human subjects is probably related to the limited doses of ethanol chosen.

Cumulative excess urinary excretion of folate in rats after repeated ethanol treatment.

The folate deficiency that is produced by chronic alcohol ingestion results from poor dietary intake and from effects of ethanol on folate metabolism and absorption. Previous studies in fasted rats showed that singular treatment with ethanol produces an increase in urinary folate levels, in amounts that account for a subsequent decrease in plasma folate levels. The effects of subacute administration of ethanol on urinary folate excretion were studied in fed and fasted rats treating for 1, 2, 3 or 4 d either with ethanol orally in four doses of 1 g/kg each at hourly intervals or with glucose in isocaloric doses. Urine was collected at timed intervals up to 12 h after each daily dose. Rats were fed daily except for the evening prior to the final treatment day. Ethanol treatment produced an increase in urinary folate excretion in fed and in fasted rats, although the effect in fed rats was less marked. The increased excretion was similar on each final day, whether rats were treated for 1, 2, 3 or 4 d, indicating that there was no adaptation to the loss of folate during this subacute treatment. The excess urinary folate excretion accumulated so that the longer the rats were exposed to ethanol, the greater the urinary loss. These results suggest that when animals are chronically treated with ethanol, urinary folate loss could contribute to the development of folate deficiency.

Acute ethanol ingestion by humans and subacute treatment of rats increase urinary folate excretion.

Previous studies with rats showed that acute treatment with ethanol (4 g/kg) produce a marked increase in urinary folate levels, followed by a decrease in plasma folate levels. Analogous studies with human volunteer subjects using a lower dose of ethanol showed that there were small, but statistically significant increases in urinary folate levels after four hours. The initial ethanol dose was 1.0 g/kg with a single supplement of 0.1-0.2 g/kg to maintain ethanol blood levels at about 100 mg/dl for six hours. Further studies with rats were designed to test the cumulative effects of repeated daily doses of ethanol. Male Sprague-Dawley rats were treated for 1, 2, 3, or 4 days either with ethanol orally in 4 doses of 1 g/kg each at 0, 1, 2, and 3 hours or with glucose orally in 4 isocaloric doses. Urine was collected at timed intervals up to 12 hours after each daily dose. The pattern of the increase in urinary folate levels was similar in all groups, whether treated for 1, 2, 3 or 4 days. These results suggest that repeated ethanol treatment can lead to a marked cumulative folate loss via increased urinary excretion and that increased urinary folate excretion may contribute to the development of folate deficiency in humans.

High pressure liquid chromatographic assay of 4-methylpyrazole. Measurements of plasma and urine levels.

4-Methylpyrazole (4-MP), a potent competitive inhibitor of alcohol dehydrogenase activity, has potential usefulness as a treatment means for methanol and ethylene glycol poisoning as well as severe disulfiram-ethanol interactions. Further study of the safety and metabolism of 4-MP in human subjects is needed before it can be used in such therapies. An HPLC assay has been developed to measure 4-MP levels in plasma and urine samples. The method was sensitive enough to quantitate 4-MP in an amount as low as 0.1 nmol. Recovery of 4-MP from spiked urine and plasma samples was greater than 90%. 4-MP levels in the plasma and urine of rats injected with an oral dose of 50 mg/kg of body weight were determined; the detectability limit in these samples was about 3 microM. The method is easy to perform and thus has practical application for research laboratories dealing with ethanol metabolism and clinical laboratories desiring to monitor 4-MP levels.

Role of ethanol metabolism in the alcohol-induced increase in urinary folate excretion in rats.

Chronic ethanol use can lead to folic acid deficiency in humans. In rats, acute doses of ethanol produce a marked increase in the urinary excretion of folate which is followed by a decrease in plasma folate levels. To assess the respective roles of ethanol and its metabolism in these effects, five groups of male Sprague-Dawley rats were treated orally as follows: (1) ethanol in four doses of 1 g/kg each at 0, 1, 2 and 3 hr; (2) ethanol as above plus the alcohol dehydrogenase inhibitor 4-methylpyrazole (4-MP) at 50 mg/kg, i.p., 15 min prior to 0 hr; (3) glucose in four isocaloric doses; (4) glucose plus 4-MP as above; and (5) methanol in four doses of 1 g/kg. Total folate levels in the urine peaked in both ethanol- and methanol-treated rats at the same time as the urine alcohol levels (after 6-8 hr) and then declined over the same time course as the alcohol levels. Concurrent administration of 4-MP inhibited the metabolism of ethanol and maintained the increase in urinary folate excretion throughout 24 hr. Ethanol administration produced minor changes in the relative distribution of folate derivatives in the urine, and these changes were not prevented by 4-MP treatment. The urinary levels of formic acid, which is metabolized by folate-dependent processes, were increased by ethanol administration; this increase was prevented by 4-MP. These results suggest that ethanol is not unique among alcohols in increasing urinary folate excretion and that ethanol metabolism plays no role in the increased urinary folate excretion. However, ethanol metabolism contributes to a second effect of ethanol on the folate system, which leads to increased urinary levels of formic acid.

Relationship of alcohol metabolism to folate deficiency produced by ethanol in the rat.

Chronic ethanol use can lead to folic acid deficiency in humans. In rats, acute doses of ethanol produce a marked increase in urinary folate excretion, which precedes a decrease in plasma folate levels. To assess the role of ethanol and its metabolism in these effects, five groups of male Sprague-Dawley rats were treated orally as follows: (1) ethanol in 4 doses of 1 g/kg each at 0, 1, 2 and 3 hr; (2) ethanol, as above, plus the alcohol dehydrogenase inhibitor 4-methylpyrazole (4-MP) at 50 mg/kg IP 15 min prior to 0 hr; (3) glucose in 4 isocaloric doses; (4) glucose plus 4-MP as above; (5) methanol in 4 doses of 1 g/kg. Urinary folate levels (by Lactobacillus casei assay) peaked in both ethanol- and methanol-treated rats at the same time as the urine alcohol levels (6-8 hr) and then declined with a similar time course. Urinary levels of formic acid, which is eliminated by oxidation by a folate-dependent pathway, were significantly increased by ethanol administration, thus indicating another ethanol-folate interaction. Concurrent administration of 4-MP suppressed the increased excretion of formate but had no effect on the increased excretion of folate in ethanol-treated rats. These studies suggest that ethanol has two distinct effects on folate metabolism, one dependent and one independent of ethanol metabolism.