Treatment of diabetes mellitus in dogs using isophane insulin penfills and the use of serum fructosamine assays to diagnose and monitor the disease.

The objectives of the study were to test the use of a prefilled insulin syringe (Insulatard Novolet, isophane insulin, 100 IU/mL) in treating diabetic dogs and to test the clinical usefulness of serum fructosamine measurements in diagnosing and monitoring diabetes mellitus in dogs. For this study 15 dogs from throughout Norway with newly diagnosed diabetes mellitus were included and treated over a period of 180 days. All 15 dogs showed pretreatment hyperglycaemia. Of the 13 dogs tested, all showed elevated pretreatment serum fructosamine values. Within 2 weeks, 3 of the 15 included dogs had dropped out of the study. In 8 of the 12 remaining dogs, the clinical signs ceased within this period. Within a month, another dog was euthanised and one had died. Seven of the 10 remaining dogs were clinically normal. Three dogs had normal serum fructosamine concentrations, while in 6 dogs moderately or highly elevated serum fructosamine concentrations persisted. In one case serum fructosamine was not measured at this time. Increase in serum fructosamine concentration seemed to reflect hyperglycaemia and deteriorated clinical condition. Decrease in serum fructosamine concentration seemed to reflect improved glycaemic status and clinical condition. During the study period the owners did a total of approximately 3500 injections on their dogs. No reports of injection difficulties were received. This study documents that Insulatard Novolet is easy and safe to use in treating diabetic dogs and that serum fructosamine reflects long-term glucose concentrations in dogs. Serum fructosamine measurements provided a simple and easy way to diagnose persistent hyperglycaemia and monitor the treatment in diabetic patients.

[Long-term therapy of true precocious puberty in girls using Decapetyl-Depot, a superactive gonadoliberin agonist in depot microcapsulated form]. / Dlouhodobá lécba dívek s pubertas praecox vera prípravkem Decapeptyl-Depot, superaktivním agonistou gonadoliberinem ve forme retardovaných mikrotobolek.

Within the framework of an international prospective multicentre study 93 girls with gonadotropin-dependent precocious puberty were treated with Decapeptyl-Depot LHRH agonist (75 micrograms/kg, once a month by the i.m. route). This treatment led to prompt, immediate and long-term suppression of the pituitary-ovarian axis with subsequent regression of premature onset of secondary sex signs, to the arrest of premature menstruation, to normalization of the pathologically accelerated growth and inhibition of the accelerated bone maturation. This improved the mean expected growth during the first four years of treatment from 159 to 165 cm (p < or = 0.01). Concurrently in the majority complete normalization of the severely impaired mental condition of the affected girls due to the premature onset of puberty occurred (mean onset of puberty -4.5 years). Selective suppression of the pubertally increased gonadotropin secretion was even after prolonged treatment fully reversible after termination of treatment with a subsequent onset of normal puberty and cyclic ovarian activity. Undesirable side-effects and antibodies against Decapeptyl were not detected, the local tolerance of the injected microcapsules was satisfactory. GnRH agonists in depot form are becoming, due to their superior suppressive action incl. that on bone maturation, the drug of choice in long-term treatment of central precocious puberty as they are the first which mitigate or can prevent, if treatment is started in time, a final small stature which frequently is the greatest handicap for a life-time (frequently associated with adverse bodily disproportions).

The use of isophane insulin for the control of diabetes mellitus in dogs.

For this study 54 dogs with diabetes mellitus verified by anamnesis, clinical examinations and laboratory analyses were selected in 13 Danish and Swedish small animal clinics. After instruction the owners gave isophane insulin ("Insulin Protaphan Human") injections to the dogs morning and evening followed by a commercial or homemade meal rich in fibers. The veterinarians examined the treated dogs 5 times or more in the 90 day treatment period, preferably in the morning before injection and meal. In all 54 dogs the clinical symptoms disappeared a few days after isophane insulin injections, and 54% of the dogs were clinically healthy within 8 days. Within a month 96% of the dogs were normalized after therapy. Simultaneously the blood glucose levels were normalized in 64% of the dogs within 14 days and in further 21% within 30 days. The urine glucose levels were normalized in 64% of the dogs within 14 days and for further 19% within 30 days. At the end of the study 48 out of the 54 diabetic dogs were clinically healthy, alert and free from symptoms of diabetes. The average dose of isophane insulin was for greater dogs 0.44 units per kg bw twice a day, for small dogs 0.79. Six dogs had been destroyed in the trial period for various reasons. One owner had injection troubles. Another owner was hospitalized and had to get rid of the dog. One dog developed advanced breast cancer, 1 went fierce and 2 developed cataracts. Four dogs had by 1 or 2 occasions shown hypoglycemic symptoms, which quickly disappeared after appropriate adjustments of insulin dosing, feeding schedule and exercise programme.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved test to identify heterozygotes for congenital adrenal hyperplasia without index case examination.

In an attempt to improve detection of heterozygote carriers of the gene for congenital adrenal hyperplasia (21-hydroxylase deficiency; CAH) 64 families with at least 1 affected member (72 homozygotes and 191 clinically healthy subjects) were studied by HLA genotyping and by the single-dose corticotropin stimulation test. Plasma samples were drawn immediately before corticotropin and 60 min after its injection, and they were analysed simultaneously for eight adrenal steroids by radioimmunoassay after extraction and automated gel chromatography. Heterozygosity was defined as the presence of one HLA haplotype in common with the affected relative. Of the various basal and corticotropin-stimulated steroid levels and their ratios, the ratio of 17-hydroxyprogesterone to 11-deoxycorticosterone after corticotropin had the greatest power to discriminate between heterozygotes and normal relatives; that ratio was significantly higher in the heterozygotes (n = 116) than in the normal relatives (n = 75) and there was no overlap between the groups (range 12.2-214 vs 1.2-11.9). Thus, it is possible to detect all CAH heterozygotes without examining the index case by means of specific steroid analysis.

Treatment of children with central precocious puberty by a slow-release gonadotropin-releasing hormone agonist.

A total of 82 patients (74 girls, 8 boys) are presently participating in an international multicentre trial for treatment of central precocious puberty (CPP) with a slow release gonadotropin-releasing hormone (GnRH) agonist depot preparation: Decapeptyl-Depot (DD). Of these patients, 53 (3 boys) were previously untreated (group 1) and 29 (5 boys) have been treated before with either a short-acting GnRH analogue or cyproterone acetate (group 2). Fifty-one patients (44 girls, 7 boys) were treated with DD for 12 months or more. Basal plasma luteinizing hormone (LH) levels decreased in both groups after 1 year of therapy. The LH response to intravenous GnRH was reduced in both groups. Basal plasma follicle stimulating hormone (FSH) levels decreased in both groups. Stimulated FSH levels were reduced in both groups after 1 year of DD treatment. Plasma oestradiol levels in the girls decreased to prepubertal levels in both groups. In all patients the clinical signs of precocious gonadarche such as breast development and menstruations (girls) and an increased testis volume (boys), did not further progress and sometimes regressed in several patients. Growth velocity decreased in the girls of group 1 from 9.0 +/- 0.72 cm/year (mean +/- SEM) in the last half-year before treatment to 6.3 +/- 0.50 in the first half-year of treatment (P less than 0.01) and to 4.5 +/- 0.23 cm/year in the second half-year (P less than 0.01). After 12 months a stabilization of growth velocity was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

[The significance and characteristics of the LHRH test in diagnosing precocious puberty development in girls: the stimulated LH/FSH quotient differentiates between central precocious puberty and premature thelarche]. / Bedeutung und Charakteristika des LHRH-Testes in der Diagnostik der vorzeitigen Pubertätsentwicklung bei Mädchen: Der stimulierte LH/FSH-Quotient differenziert zwischen zentraler Pubertas praecox und prämaturer Thelarche.

LHRH tests (100 micrograms i.v.) were performed in 31 girls with central precocious puberty (PP); the girls were participating in an international multicentre trial for the treatment of PP with the LHRH agonist decapeptyl in microspheres, together with 18 girls with premature thelarche (PT). Assignment to these two groups was made after 6 months to 5 years of clinical follow-up. LH and FSH were determined centrally using a polyclonal RIA. Basal LH and FSH levels and stimulated LH levels were significantly higher in PP patients (p less than 0.001), but the stimulated FSH levels were not significantly different between the two groups. In the PP group, all stimulated LH levels were above the prepubertal range, whereas in the PT patients all stimulated LH levels were within the prepubertal reference limits. In PP and PT patients 52% and 56%, respectively, of the stimulated FSH levels were increased above the range for prepubertal girls. In 55% of the PP patients, stimulated LH levels were also above the reference range for the corresponding Tanner breast stage. In contrast, all stimulated LH levels of the PT group were within the reference limits for their breast stage. For FSH, 45% and 56% of the stimulated levels were above the normal ranges for the corresponding breast stages in the PP and PT groups, respectively. The LH-to-FSH ratio after LHRH stimulation was significantly higher in the PP than in the PT group (p less than 0.001). All but one of these ratios were above 1 in the PP patients and all ratios in the PT patients were below 1.(ABSTRACT TRUNCATED AT 250 WORDS)

HLA-A,B,C,DR typing and 17-OHP determination for second trimester prenatal diagnosis of 21-hydroxylase deficient CAH.

In 18 families at risk for the HLA-linked, 21-hydroxylase deficient form of autosomal recessive congenital adrenal hyperplasia (CAH), prenatal diagnosis (PD) was performed using two methods: (1) HLA-A,B,C typing and in the latter 11 cases also DR typing of cultured amniotic fluid cells (AFC) using the standard microcytotoxicity assay, and (2) measurement of second trimester amniotic fluid 17-hydroxyprogesterone (17-OHP) concentration using gel chromatography and radioimmunoassay. The accuracy of the prenatal predictions was confirmed by postnatal HLA typing of umbilical cord blood lymphocytes and by clinical evaluation. In 16/18 families, both HLA typing of AFC and 17-OHP measurements proved informative for PD. The predictions of both methods were concordant in 14/16 families (88 per cent). In ten of these families, a normal fetus was predicted, and in four, an affected fetus; all pregnancies were carried to term and all predictions were confirmed postnatally. In 2/16 cases (12 per cent), however, the predictions were discordant: the prenatal HLA typing indicated an affected fetus, whereas the 17-OHP values predicted a normal fetus. Both pregnancies were continued and two healthy boys were delivered. The discordance proved to be due to a 'missed' HLA antigen in one case and to serologically cross-reactive HLA antigens in the second. Finally, in 2/18 cases, prenatal assessment of fetal genotype had to rely on HLA typing alone as 17-OHP measurement was not performed in one family and in the second family the 17-OHP values obtained were not informative due to inadvertent continuation of hormone therapy to the date of amniocentesis. In both cases, the HLA typing data accurately predicted a normal fetus. In conclusion, a combination of HLA typing of cultured AFC and 17-OHP measurements of amniotic fluid permits accurate prenatal diagnosis of CAH in most cases (88 per cent). In addition, the supplementary use of HLA-DR typing of AFC as presented here for the first time proved helpful in families with HLA-A,B homozygosity due to parental sharing of antigens and can be informative for identifying HLA-B/21-OH recombinant haplotypes.

Genotyping steroid 21-hydroxylase deficiency: hormonal reference data.

Hormonal reference data, in the form of nomograms relating baseline and stimulated levels of adrenal hormones, provide a means of genotyping steroid 21-hydroxylase (21-OH) deficiency in congenital adrenal hyperplasia. Data from both 360- and 60-min ACTH stimulation tests are given. The serum hormone concentrations that have proven most useful in classifying 21-OH deficiency are 17-hydroxyprogesterone and delta 4-androstenedione. These nomograms clearly distinguish the patient with classical 21-OH deficiency from those with the milder symptomatic and asymptomatic nonclassical forms of 21-OH deficiency (previously referred to as late onset and cryptic forms) as well as heterozygotes for all of the forms and those subjects predicted by HLA genotyping to be unaffected. The nomograms also can identify individuals heterozygous for 21-OH deficiency in the general population who have a characteristic heterozygote response. These nomograms provide a powerful tool by which to assign the 21-OH deficiency genotype. Patients whose hormonal values fall on the regression line within a defined group are assigned to that group. In view of the strong correlation between the 60- and 360-min ACTH stimulation tests, the less cumbersome and shorter 60-min test can be used with the same confidence as the longer test.

Preclinical studies on human insulin. I. Acute and subacute toxicity studies.

Human insulin (prepared from porcine insulin) and porcine insulin were tested for acute toxicity by subcutaneous administration to unfasted mice and rats and fasted mice. The animals were observed for signs of reaction and post mortem examination was performed. LD50 values were calculated when possible. The LD50 values in the unfasted animals were several times higher than in the fasted mice, but similar for the two insulin preparations. The deaths and the signs observed were most probably caused by hypoglycaemia. No dose-related macroscopic organ changes were found. In a 28 day toxicity study rats were given human or porcine insulin subcutaneously at dosages of 2, 20 or 200 U/kg/day. The criteria examined included mortality, body-weight change, food consumption and utilization, haematology, blood chemistry, urinalysis, ophthalmoscopy, organ weights, gross- and histopathology. A few deaths occurred because of hypoglycaemia. In surviving rats from dosage groups 20 and 200 U/kg/day of either insulin preparation higher plasma glucose levels than in the control were observed 24 hours after dosing. Higher food intake and body-weight gains, lower plasma protein concentrations and higher urinary volumes with associated low specific gravity were observed in animals given either human or porcine insulin at 200 U/kg/day. No other adverse effects were registered, and no overt difference was found between the effect of human and porcine insulin.

Late-onset steroid 21-hydroxylase deficiency: a variant of classical congenital adrenal hyperplasia.

Hormonal studies and human leukocyte antigen (HLA) genotyping were performed in 5 males and 13 females who were demonstrated to have 21-hydroxylase deficiency. The enzymatic deficiency of steroidogenesis was detected by family studies of 10 females who presented with varying symptoms of androgen excess. The 10 index cases had normal genitalia at birth, but virilized to varying degrees postnatally. The additional 8 affected family members had not sought medical care, but some were found to have signs of virilization on physical examination, while others were normal. Thus both late-onset (symptomatic) and cryptic asymptomatic) 21-hydroxylase deficiency occurred in the same pedigree. The hormonal and genetic linkage studies indicate that the late-onset (symptomatic) form of 21-hydroxylase deficiency, like the cryptic (asymptomatic) and classical forms of 21-hydroxylase deficiency, is transmitted by an autosomal recessive gene which is linked to HLA-B. Furthermore, the classical form of 21-hydroxylase deficiency associated with prenatal virilization is transmitted by an allelic variant for steroid 21-hydroxylase different from that of the nonclassical forms, late-onset (symptomatic) and cryptic (asymptomatic) 21-hydroxylase deficiency. Although these latter 2 disorders have different clinical manifestations, they demonstrate a similar degree of steroid 21-hydroxylase deficiency that is less severe than that observed in classical 21-hydroxylase deficiency. The hormonal and genetic linkage data indicate that cryptic (asymptomatic) and late-onset (symptomatic) 21-hydroxylase deficiency result from the same allelic variant at the steroid 21-hydroxylase locus. A glossary of terms is presented to describe the various allelic forms of 21-hydroxylase deficiency with consistency.

Genetic and hormonal characterization of cryptic 21-hydroxylase deficiency.

Cryptic 21-hydroxylase deficiency has been previously described in asymptomatic family members of patients with classical congenital adrenal hyperplasia (CAH). These family members were detected by high baseline 17-hydroxyprogesterone levels found in the course of family studies. The hormonal responses to ACTH of the family members with cryptic 21-hydroxylase deficiency were determined and compared to the responses of patients with CAH, patients with acquired adrenal hyperplasia, family members predicted to be heterozygous for CAH, family members predicted to be unaffected, and the general population. The ACTH-stimulated levels of 17-hydroxyprogesterone and delta 4-androstenedione in the cryptic family members were elevated above the level of the general population or family members heterozygous for classical CAH, but below that of patients with CAH. The hormonal profile of patients with cryptic 21-hydroxylase deficiency is similar to that of patients with acquired adrenal hyperplasia. The response of family members heterozygous for the cryptic gene (21-OH CRYPTIC/21-OH NORMAL) was indistinguishable from that of family members heterozygous for the classical CAH gene (21-OH CAH/21-OH NORMAL). These studies support our previous proposal that patients with cryptic 21-hydroxylase deficiency are genetic compounds, having one gene for a severe enzyme deficiency and one gene for a mild 21-hydroxylase deficiency. Thus, the 21-hydroxylase genotype in cryptic 21-hydroxylase deficiency is 21-OH CAH/21-OH CRYPTIC.

HLA linkage and B14, DR1, BfS haplotype association with the genes for late onset and cryptic 21-hydroxylase deficiency.

Classical congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21-OH-def) has been established to be an HLA-linked, recessive monogenetic disease. However, two nonclassical forms of 21-OH-def have also been described: "cryptic" 21-OH-def, which has been shown to be HLA-linked, and "late onset" 21-OH-def, for which the status of linkage to HLA has been less certain. We now describe studies of eight additional unrelated probands with symptomatic, "late onset" 21-OH-def, and conclude that this form is also HLA-linked. Both "late onset" and "cryptic" 21-OH-def are highly associated with the same HLA antigens and markers (HLA-B14, HLA-DR1, and Bf type S) in individuals from different ethnic and geographical backgrounds. Since both "late onset" and "cryptic" 21-OH-def appear to occur in individuals with one classical 21-OH-def (21-OHCAH) allele who in addition have another 21-OH-def allele, as well as in individuals who appear to be homozygous for variant 21-PH-def alleles, and since both late onset and cryptic 21-OH-def appear to occur in the same families, our data suggest that these syndromes may represent different clinical expressions of similar or identical nonclassical 21-OH-def alleles.

Cryptic 21-hydroxylase deficiency in families of patients with classical congenital adrenal hyperplasia.

Serum androgens and 17-hydroxyprogesterone concentrations and HLA genotypes were determined in 124 families of patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH). In 8 pedigrees, we discovered 16 pubertal or postpubertal family members of either sex who had biochemical evidence of 21-hydroxylase deficiency but were without clinical symptoms of excess virilism, amenorrhea, or infertility. We designated these family members as individuals with cryptic 21-hydroxylase deficiency. Within each generation, the family members with cryptic 21-hydroxylase deficiency were HLA identical. It is proposed that these family members are genetic compounds, having 21-hydroxylase deficiency as a result of two recessive gene defects: 1) a severe 21-hydroxylase gene defect present in the index case with classical CAH (21-OHCAH) and 2) a mild 21-hydroxylase gene defect (21-OHCRYPTIC). Thus, the CAH genotype in the family members with cryptic 21-hydroxylase deficiency is 21-OHCAH/21-OHCRYPTIC. Lod score analysis for linkage between the cryptogenic 21-OH trait and HLA gave a combined Lod score for males and females of theta = 0.00 of 3.409. Close genetic linkage between HLA and 21-OHCRYPTIC was thus established. This study provides support for the previously reported heterogeneity of 21-hydroxylase deficiency which may result from allelic variability at the locus for steroid 21-hydroxylase.

Hormonal studies in obligate heterozygotes and siblings of patients with 11 beta-hydroxylase deficiency congenital adrenal hyperplasia.

Hormonal response to ACTH stimulation and HLA genotyping were determined in families of patients with 11 beta-hydroxylase deficiency congenital adrenal hyperplasia. Neither hormonal measurements nor HLA genotyping were useful for the detection of heterozygosity in the families.

Hormonal phenotype and HLA-genotype in families of patients with congenital adrenal hyperplasia (21-hydroxylase deficiency).

The response of 17-hydroxyprogesterone (17-OHP) and cortisol (F) to a 6-hr ACTH stimulation in families of patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency was studied. These studies demonstrated that siblings who should be heterozygous carriers of the 21-hydroxylase deficiency gene based on HLA genotyping are hormonally different from the general population. In pre- and early pubertal children predicted to be heterozygous carriers of the gene based on HLA genotyping, the 17-OHP level (13.1 +/- 4.5 ng/ml), the rate of increase of 17-OHP (0.03 +/- 0.01), and the ratio of 17-OHP/F at 6 hr (0.27 +/- 0.07) were significantly higher (P less than 0.001) than in the control population, (3.9 +/- 1.9, 0.009 +/- 0.005, and 0.08 +/- 0.04 ng/ml, respectively). In late and postpubertal males, these hormonal parameters in the heterozygotes (17 +/- 9.7, 0.04 +/- 0.026, 0.42 +/- 0.33 ng/ml, respectively) were significantly higher (P less than 0.001) than in the general population (5.3 +/- 1.6, 0.009 +/- 0.004, and 0.1 +/- 0.03 ng/ml, respectively). In postmenarchal females, the mean hormone responses in the heterozygotes (12.1 +/- 9.7, 0.03 +/- 0.02, and 0.27 +/- 0.24 ng/ml, respectively) were significantly higher (P less than 0.005, less than 0.01, less than 0.005, respectively) than in the general population (5.2 +/- 2.5, 0.01 +/- 0.007, and 0.1 +/- 0.04 ng/ml, respectively). However, the overlapping values did not permit a clear differentiation of the hormonal responses in these two groups. Another (ACTH) stimulation in one family demonstrated that a father of a patient probably is a previously unrecognized homozygous affected patient and,thus, revision of the congenital adrenal hyperplasia (CAH) genotype for this family was required.