Liver function tests in diabetic patients.

Nine different liver function tests (LFT) were assessed in 175 unselected diabetic outpatients stabilized on diet, insulin, or oral hypoglycemic drugs. In another group of 72 diabetic inpatients having diagnostic liver biopsy, relationships between LFT and histologic changes in the liver were investigated. Abnormalities in at least one of the tests were noted in 57% of the outpatients, and two tests gave pathologic results in 27%. The non-insulin-dependent diabetic patients more often had abnormal LFT results than did the insulin-dependent diabetic patients. Serum chenodeoxycholic acid concentrations were increased in 27%, gamma-glutamyl transpeptidase (gGT) activities in 19%, and alanine aminotransferase (Alt) activities in 17% of the outpatients, but the increases were rarely more than twice the upper limit of normal. In multivariate analysis, outpatients who were overweight, showed poor diabetes control during a short duration of diabetes controlled by treatment with diet or oral agents, and had a mature age at onset of diabetes displayed the most significant clinical explanatory variables associated with abnormal Alt. In the inpatients, the percentages of abnormal Alt and gGT results were augmented, along with increasing severity of histologic changes, but the mean values of Alt and gGT did not differ significantly between the various histologic groups. In addition, the diabetic patients with nonspecific inflammatory changes or increase in liver fibrosis often showed normal or only minor elevations in these test values.

The effects of an antiprogestin, mifepristone, and an antiestrogen, tamoxifen, on endometrial 17 beta-hydroxysteroid dehydrogenase and progestin and estrogen receptors during the luteal phase of the menstrual cycle: an immunohistochemical study.

The effects of a progesterone antagonist, mifepristone (RU486), and an estrogen antagonist, tamoxifen, given during the early luteal phase on endometrial 17 beta-hydroxysteroid dehydrogenase (17HSD) and estrogen (ER) and progesterone (PR) receptors were studied. Eleven regularly menstruating women were studied during control and treatment cycles. In the treatment cycle on day LH + 2 (2 days after the peak serum LH concentration), 10 subjects received a single dose of 200 mg mifepristone, and 9 received 2 doses of 40 mg tamoxifen on days LH + 2 and LH + 3. In addition, 4 subjects received 400 mg mifepristone in a separate treatment cycle. 17HSD, ER, and PR were measured immunohistochemically in endometrial tissue specimens taken on days LH + 6 to LH + 8. Blood samples were conducted during control and treatment cycles, and serum estradiol, progesterone, and LH concentrations were quantified by RIA. Administration of mifepristone blocked the induction of 17HSD by progesterone and prevented the expression of 17HSD in gland and surface epithelial cells in 8 patients. In 2 patients, staining of 17HSD was seen during both the control and mifepristone treatment cycles. The higher dose of mifepristone additionally given to four subjects did not block the expression of 17HSD in 2 cases where blocking was observed with the lower dose of mifepristone, and in 1 of these patients, very strong staining of 17HSD was observed in basal cells beneath the epithelial cells. ER and PR showed intense staining in the nuclei of both gland and stromal cells in mifepristone treatment cycles, whereas receptor staining was faint or absent in the respective control cycles. Tamoxifen did not have any significant effect on staining of 17HSD or the abundance of receptors. Serum concentrations of estradiol, progesterone, and LH were not significantly affected by the administration of mifepristone or tamoxifen. This study reveals that mifepristone, administered in the early luteal phase, usually blocks the expression of 17HSD and the down-regulation of PR and ER. However, the expression of 17HSD in some patients may reflect the ineffectiveness of the mifepristone treatment used to prevent implantation in certain subjects.

Complete amino acid sequence of human placental 17 beta-hydroxysteroid dehydrogenase deduced from cDNA.

cDNA clones for 17 beta-hydroxysteroid dehydrogenase (17-HSD; EC 1.1.1.62) were isolated from a placental lambda gt11 expression library using polyclonal antibodies against placental 17-HSD. The largest cDNA contained 1325 nucleotides, consisting of a short 5'-noncoding segment, a coding segment of 987 nucleotides terminated by a TAA codon, and a 329 nucleotide long 3'-noncoding segment. The open reading frame encoded a polypeptide of 327 amino acid residues with a predicted Mr of 34853. The amino acid sequence of 23 N-terminal amino acids determined from purified 17-HSD agreed with the sequence deduced from cDNA. The deduced amino acid sequence also contained two peptides previously characterized from the proposed catalytic area of placental 17-HSD.

Immunohistochemical detection of human estrogen receptor with region D-specific antipeptide antibodies.

To study the possibility of using antipeptide antibodies for the immunohistochemical determination of human estrogen receptors (ER), three peptides corresponding to the putative major antigenic regions of the human ER (Met12-Leu26, or ERP1; Thr227-Gln267, or ERP2; Leu256-Gly275, or ERP3) were used to produce site-specific rabbit polyclonal antipeptide antisera. High titer antibodies were obtained against all the peptides used, as judged by time-resolved fluoroimmunoassay. The antibodies against region D (ERP3) specifically immunoprecipitated the ER proteins in vitro, as did the antiERP2 antibodies to a much smaller extent. With one of the region D-specific antibodies (antiERP3 Ab2) ER could also be immunohistochemically detected. When benign and malignant human breast and normal endometrial tissues were used, the immunohistochemical staining observed with these antipeptide antibodies correlated well with the staining obtained with an established method. Thus, the results reported here show that this part of region D in ER is a potential antigenic epitope for the production of site-specific antibodies against ER. Antipeptide antibodies produced against this region can be used to immunolocalize the ER in various normal and pathological human tissues.

Amniotic fluid bile acids in normal and pathologic pregnancy.

Radioimmunologic techniques were used to determine 2 primary bile acids (cholic and chenodeoxycholic acid) and 1 secondary bile acid (deoxycholic acid) from human amniotic fluid of healthy pregnant women and from patients with diabetes, toxemia, or intrahepatic cholestasis during pregnancy. In general, the mean bile acid concentrations in the amniotic fluid were very similar to those in the serum, although in paired samples from individual patients these 2 values did not correlate significantly. Very high levels of the 2 primary bile acids were measured from the amniotic fluid of patients with intrahepatic cholestasis. The mean values were about 70 times higher than those in the controls. Amniotic fluid cholic acid content was slightly elevated in diabetic and toxemic patients, too. Deoxycholic acid was consistently found in the amniotic fluid specimens, but there was no change in its concentration among the various groups. In this limited series of patients, no significant correlation was found between the bile acid concentrations in the amniotic fluid and signs of fetal distress at the time of amniocentesis, although the lowest maternal serum estriol and human placental lactogen values were associated with the highest amniotic fluid bile acid concentrations. The condition of the newborn infants did not correlate with amniotic fluid bile acid concentrations in any of the patient groups studied. It thus appears that high amniotic fluid bile acid content present a threat to the fetus, but further studies are needed to clarify this point.

Sex steroid hormones and receptors in relation to S-phase fraction and ploidy level in endometrial carcinoma.

Although endogenous hormones exert an effect on the proliferation of endometria adenocarcinoma, there also seems to be an autonomous proliferation of the malignant cells. Simultaneous measurement of endocrine and cell proliferation related variables in endometrial adenocarcinoma specimens are expected to increase the understanding of factors responsible for progression or regression of this form of cancer. Sixty patients with endometrial adenocarcinoma were examined. The following parameters were analysed: endogenous plasma concentration of oestradiol, oestrone, progesterone, androstenedione and testosterone; S-phase fraction (SPF) and ploidy level, by flow cytometry; oestrogen and progesterone receptors, by immunohistochemistry. The oestrogen receptor positive tumours had a lower S-phase fraction that receptor negative tumours (p < 0.05), but SPF was still under the mean for the whole group. ER positive tumours were all diploid, while progesterone receptors were found also in aneuploid tumours. The presence of PR did not relate to lower SPF, but in an earlier study increased progesterone concentration was found to relate to lower SPF, and the antiproliferative effect of progesterone was also seen in more malignant tumours.

Serum bile acids and lipids during treatment of climacteric symptoms with natural oestrogen--progestin combinations.

Climacteric symptoms of 21 women were treated for 6 mth with sequential combination preparations containing natural oestrogen (oestradiol and oestriol) and norethisterone acetate as progestin. There were no significant changes during the treatment period in the serum alanine aminotransferase activity or concentrations of cholesterol, high-density lipoprotein (HDL)-cholesterol, triglycerides, cholic acid and deoxycholic acid. The concentration of chenodeoxycholic acid was, however, significantly decreased after 6 mth treatment. It thus appears that the above natural oestrogen--progestin combinations do not have adverse effects on hepatic function and lipid metabolism.

Serum bile acids during biphasic contraceptive treatment with ethinyl estradiol and norgestrel.

Twenty-nine women were treated with biphasic combined oral contraceptive pills containing ethinyl estradiol 0.05 mg and levonorgestrel 0.050-0.125 mg. Serum primary bile acids (cholic acid and chenodeoxycholic acid) and one secondary bile acid (deoxycholic acid) were measured by radioimmunoassay. The serum samples were collected before the treatment and at one, three and twelve months during the treatment. No significant changes were found in these bile acid levels during the treatment. The ratio of cholic/chenodeoxycholic acid did not change either. No pathological values were found in the conventional liver function tests although serum alanine aminotransferase activity was significantly increased after twelve months' treatment. It can therefore be concluded that the present contraceptive pill does not cause any liver dysfunction detectable by bile acid measurements of other "classical" liver function tests.

Gallbladder function and maternal bile acids in intrahepatic cholestasis of pregnancy.

Ultrasonic measurement of the gallbladder volume was taken in 8 nonpregnant, healthy women, in 7 women with normal pregnancies and in 7 women whose pregnancies were complicated by intrahepatic cholestasis of pregnancy, in the fasting state and 30, 60, 120 and 180 min after a test meal. At the same time the serum concentrations of cholic acid and chenodeoxycholic acid were measured. The fasting and ejection volumes of the gallbladder in cholestasis of pregnancy were greater than in normal pregnancy. The fasting volume of the gallbladder was greater, but the ejection volume smaller in normal pregnancy than in nonpregnant women. No difference in the ultrasonic appearance of the intra- and extrahepatic bile ducts was found between the groups. Serum bile acids were increased in cholestasis of pregnancy and did not display any decreasing tendency after the postprandial rise during the following 3 h. The results indicate that in cholestasis of pregnancy the gallbladder function and the enterohepatic circulation of bile acids are different from normal pregnancy. This may be associated with the great tendency to gallstones in these women. The large size of the gallbladder in cholestasis of pregnancy has differential diagnostic importance in the ultrasonic evaluation of a pregnant woman with liver disease.

Serum bile acid levels in intrahepatic cholestasis of pregnancy during treatment with phenobarbital or cholestyramine.

Nineteen patients suffering from the intrahepatic cholestasis (IHC) of pregnancy were studied. Twelve of them were treated with phenobarbital (100 mg/day) and seven with cholestyramine (18 g/day). The overnight fasting levels of serum cholic acid (CA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) were measured by radioimmunoassay. The activities of serum transaminases, gamma-glutamyltranspeptidase, alkaline phosphatase and total and conjugated bilirubins were also analyzed. It was found that there was no correlation between the itching symptom and the serum bile acid levels. During phenobarbital treatment serum bile acid concentrations did not change. Also, the other measured parameters as well as the CA/CDCA ratio did not change significantly. Transaminases had, however, a slight tendency to decrease. The therapy successfully relieved itching in half of the cases. There was no relationship between the relief of the itching and the change in the bile acid concentrations. Cholestyramine treatment did not decrease the CA level significantly, but that of the CDCA decreased (P less than 0.05) and the ratio of CA/CDCA increased (P less than 0.05). In the other analyzed liver function test results, an increase (P less than 0.05) occurred only in the concentrations of conjugated bilirubin. The itching was relieved in five of the seven cases during the first week of treatment, but after that the symptom tended to reappear. There was a slight correlation between the decrease in the CDCA level and in the relief of the itching. The two drugs did not cause any particular side effects.

Radioimmunoassay of conjugated cholic acid, chenodeoxycholic acid, and deoxycholic acid from human serum, with use of 125I-labeled ligands.

We describe a method for radioimmunoassay of conjugated cholic acid, chenodeoxycholic acid, and deoxycholic acid in serum. In the method, 125I-labeled bile acid conjugates are used as the tracers along with antibodies raised against individual bile acid-bovine serum albumin conjugates. Antibody-bound and free bile acids were separated by polyethylene glycol precipitation (final concentration, 125 g/L). Before radioimmunoassay, 0.1-mL serum samples were precipitated with nine volumes of ethanol, and portions from the supernate were used in the assays. The lowest measurable amounts of the bile acids, expressed as pmol/tube, were: cholic acid conjugates, 2; chenodeoxycholic acid conjugates, 0.5; and deoxycholic acid conjugates. 2. Analytical recovery of bile acids added to bile acid-free serum ranged from 85 to 110%; intra-assay and inter-assay CVs ranged from 3.2 to 5.3% and from 5.3 to 12.2%, respectively. Concentrations (mean +/- SD) of the bile acid conjugates in serum from apparently healthy women and men (in mumol/L) were: cholic acid conjugates, 0.43 +/- 0.17 (n = 126); chenodeoxycholic acid conjugates, 0.47 +/- 0.23 (n = 111); and deoxycholic acid conjugates, 0.33 +/- 0.11 (n = 96). The values for primary bile acids were greatly increased in patients with various hepatobiliary diseases.

Time-resolved immunofluorometric assay of 17 beta-hydroxysteroid dehydrogenase in plasma.

We describe a time-resolved immunofluorometric assay (TR-IFMA) for human 17 beta-hydroxysteroid dehydrogenase (17HSD) in which antibody-coated microtiter strip wells and europium chelate-labeled polyclonal antibodies are used. In preparing the label, a polyclonal antibody is affinity-purified and derivatized with diethylenetriamine-pentaacetic acid. With this derivative, five to eight europium ions can be combined with one antibody molecule without decreasing the antibody's immunoreactivity. The minimum detectable concentration of 17HSD is 0.13 microgram/L; the intra- and interassay CVs are less than 8% and less than 15%, respectively, for concentrations between 0.3 and 100 micrograms/L. There is no difference between the concentrations of 17HSD in plasma specimens taken during the proliferative and luteal phases of the menstrual cycle, the measured mean concentration being 0.22 microgram/L. We found no correlation between plasma 17HSD and progesterone concentrations. The plasma concentrations of 17HSD increase during pregnancy, the mean concentrations being 1.5, 4.4, and 12.5 micrograms/L, during the first, second, and third trimesters of pregnancy, respectively. In the specimens from 18 men, the mean concentration was 0.18 microgram/L. In six plasma specimens from patients with endometrial adenocarcinoma, the mean concentration was 0.20 micrograms/L. Pre-analytical aspects are important in the assay of 17HSD because of the lability of the enzyme protein. Preferably, blood should be sampled into EDTA-containing tubes, plasma should be separated within 15 min, and glycerol must be added without delay to a final volume of 200 mL/L.

Direct solid-phase time-resolved fluoroimmunoassay of 17 alpha-hydroxyprogesterone in serum and dried blood spots on filter paper.

We describe a direct, solid-phase time-resolved fluoroimmunoassay (TRFIA) for measuring 17 alpha-hydroxyprogesterone (17OHP) in serum and blood spots on filter paper. We used 17OHP-3-carboxymethyloxime (17OHP3CMO) coupled to polylysine as the label, which enabled incorporation of up to 34 atoms of europium per molecule of 17OHP, for a very high specific activity. The assay is based on competition between labeled 17OHP3CMO and 17OHP in blood specimens for polyclonal rabbit anti-17OHP antibodies. The antibody-label complex is separated by binding to anti-rabbit antibodies coated onto microtiter strips. The assay buffer contains danazol to displace 17OHP from steroid-binding proteins in serum. For serum samples, the assay is accomplished in 1 h of incubation at room temperature. The blood spot assay with filter paper discs involves incubation overnight at 4 degrees C. Results for both types of specimens from the same subjects correlated well. The lowest measurable concentrations of 17OHP (nmol/L) were 0.10 (3 SD) and 0.75 (3 SD) for serum and dried blood on filter paper, respectively. Intra- and interassay CVs were about 5-15% for both types of samples.

Distribution of estradiol-17 beta hydroxysteroid oxidoreductase in the urogenital tract of control and neonatally estrogenized male mice: immunohistochemical, enzymehistochemical, and biochemical study.

The present study was conducted to investigate the regional distribution of the enzymes catalyzing the interconversion of the hydroxyl and carbonyl groups at C-17 of the estrogen molecule within the male urogenital tract of adult mouse and to test the hypothesis, whether regional differences in the distribution are critical for estrogen responses. The highest ratios of NADPH-dependent 3H-estrogen reduction to oxidation at C-17 of cell-free homogenates were obtained from coagulating gland and seminal vesicle as well as from the prostatic and lower intrapelvic urethra, which are considered the most estrogen-sensitive parts of the male urogenital tract. Both NADP- and NAD-dependent oxidation of 3H-17 beta-estradiol were low or nondetectable at these sites. The epithelium of the lower and prostatic urethra as well as the periurethral collecting ducts were stained with the antibody prepared against human placental 17 beta-hydroxysteroid oxidoreductase. The NAD-dependent 3H-estradiol-17 beta oxidase activity was highest in the bladder epithelium, and the activity declined sharply in the urinary tract from the bladder downward. The lowest detectable activities were found in vas deferens and prostate (combined ventral and dorsolateral lobes). The uneven distribution of estradiol-17 beta oxidase activity may provide additional explanation for the regional differences of estrogen responses. The NADPH-dependent 17 beta-reduction of estrone and the immunohistochemical staining of the human placental estradiol-17 beta oxidoreductase antigenicity were not significantly altered after neonatal estrogenization. These findings do not lend any support to the idea that the increased estrogen sensitivity observed after neonatal estrogenization is associated with changes in 17 beta-oxidoreduction. However, the possibility remains that there are specific sites (e.g., epithelium of prostatic urethra and collecting ducts) in which the changes in 17 beta-oxidoreduction of estrogen does play a role in the regulation of estrogen action.

Changes in serum bile acid concentrations during normal pregnancy, in patients with intrahepatic cholestasis of pregnancy and in pregnant women with itching.

Two primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA), and one secondary bile acid, deoxycholic acid (DCA), were measured by radioimmunoassay in pregnancy serum from 30 healthy women, 49 women with itching and 45 with intrahepatic cholestasis of pregnancy. All subjects were studied serially from between 16 and 20 weeks gestation until 35-60 days post partum. In healthy pregnant women, serum CA and DCA levels did not change significantly at any time. The mean CDCA level rose significantly towards term. In women with intrahepatic cholestasis, serum levels of CA and CDC were increased ten- and five-fold, respectively, at the time of appearance of clinical symptoms and the CA/CDCA ratio rose from 1/1 to 2/1; there was also a moderate increase in the serum concentration of DCA. In 4 of 8 women studied prospectively an increase in serum bile acid levels preceded the appearance of symptoms or other laboratory evidence of intrahepatic cholestasis. Nine of the women with itching with normal routine liver function test results had increases in serum CA and CDCA concentrations suggesting mild cholestasis.

Campylobacter-like organisms and gastritis: histopathology, bile reflux, and gastric fluid composition.

We studied a prospective series of 107 randomly chosen dyspepsia patients without gastric ulcer for the association of spiral Campylobacter-like organisms (CLO) with features of antral and fundal gastritis and duodenogastric reflux. CLO were observed in 38% of the patients. The scores for all classes of inflammatory cells in both antral and body mucosa were significantly higher in the CLO-positive patients than in the CLO-negative ones (p less than 0.001), and foveolar hyperplasia was also associated with CLO (p less than 0.05). Metaplasia and glandular atrophy in the antral mucosa were significantly commoner in the CLO-positive group (p less than 0.05 and p less than 0.01, respectively). The body gastritis score correlated significantly with age in the CLO-negative patients (R = 0.33, p less than 0.01) but not in the CLO-positive ones. There were no significant differences between the groups with regard to duodenogastric reflux or intragastric pH. The results confirm that CLO are associated with gastritis, most notably superficial gastritis in the body and atrophic gastritis in the antrum, but their aetiological significance remains to be proved.

Time-resolved immunofluorometric assay of sex-hormone binding globulin.

A time-resolved immunofluorometric assay (trlFMA) for human sex-hormone binding globulin (SHBG) is described in which antibody-coated tubes or microliter strip-wells and a europium (Eu) chelate-labeled monoclonal antibody are used. The trlFMA sensitivity is similar to that of other SHBG immunoassays, and other analytical variables compare favorably with an SHBG immunoradiometric assay (IRMA) kit and a steroid binding capacity assay: the interassay coefficient of variation (CV) is less than 8% and the intra-assay CV is less than 6% for concentrations between 6 and 200 nmol/L. The reference intervals (means +/- SD) for SHBG concentrations (nmol/L) in serum from 10 men, 10 women, and 10 pregnant women were 23 +/- 12, 65 +/- 39, and 439 +/- 122, respectively. In 14 hirsute women the mean +/- SD serum SHBG concentration (37 +/- 21 nmol/L) was significantly lower (P less than 0.01) than the mean for an age-matched, nonhirsute female comparison group. The trlFMA is technically simple, requires no centrifugation or separation reagent, and takes a counting time of only 1 s. In addition, the Eu-label is nontoxic, presents no waste-disposal problems, and has a long shelf life.

Rapid equilibrium radioimmunoassay for the amino-terminal propeptide of human type III procollagen.

This is an equilibrium-type radioimmunoassay for the amino-terminal propeptide of type III procollagen (PIIINP), which overcomes the problem of nonparallelism between the standard and human serum samples encountered with earlier assays. Proper selection of antiserum and reaction conditions diminishes interference from degradation products of the propeptide in serum. Because a rapid solid-phase-bound second-antibody step is included, the assay takes only 3 h. The intra-assay and the interassay CVs are both about 5%. In infants and children the concentration of PIIINP in serum closely parallels the growth-velocity curve. For 88 presumably healthy adults, the PIIINP concentration was 1.7-4.2 micrograms/L, about a third that measured with the previously available commercial assay. This is because of lack of inhibition by small Col 1 domain-related degradation products.