Aging reverts to juvenile conditions the synaptic connectivity of cerebral cortical pyramidal shafts. Quantitative analysis of the total number and distribution of dendritic spines along the apical shafts of layer V cerebral cortical pyramids has been performed on aging rats (90-120 to 1,135 days old) and on rats during the period of early and late development (10-80 days). As expected from previous work, present results show that the total number of dendritic spines along the shafts increase from 10 to 80 days, after which it starts to gradually decrease until the last age studied (1,135 days). The quantitative analysis of the effect of aging on the relative decrease of dendritic spines shows that this decrease starts being homogeneous along the whole length of the apical shafts and that from a certain age onwards, estimated according to present results in 400 days, this effect is significantly more pronounced in layers IV and III-II than in deep layers. Furthermore, the comparison made between the distribution of dendritic spines along the apical shafts of pyramidal neurons of old and young animals has shown that aging produces a regression of this distribution to juvenile conditions.


Effect of cocaine, ethanol or nicotine on ornithine decarboxylase activity in early chick embryo brain. Fetal drug exposure causes multiple deficits in the developing child. For both humans and animal models, the single most common drug-related problem is fetal growth suppression. This defect is associated with significant perinatal morbidity and mortality and may also be related to significant behavioral problems appearing later in life. Studies focussed on the molecular mechanism of fetal drug effects in placental models are complicated by multiple interactions of the drug with mother, placenta and fetus. Using early (76-168 h) chick embryos as a non-placental model, and three common drugs of abuse (nicotine, ethanol and cocaine) it was found that each drug suppressed the peak in fetal brain ornithine decarboxylase (ODC) activity that normally occurs at 120 h of development. For each drug, the decrease in ODC activity at 120 h was followed by a small but significant increase in ODC. Thus, although the drug-treated embryos were smaller in size, they appeared to be undergoing compensatory growth and, in fact, became equal in weight to the vehicle-treated animals, if allowed to hatch.


Development of cerebral arterial innervation: synchronous development of neuropeptide Y (NPY)- and vasoactive intestinal polypeptide (VIP)-containing fibers and some observations on growth cones. The pre- and postnatal development of sympathetic fibers containing neuropeptide Y (NPY) and parasympathetic fibers containing vasoactive intestinal polypeptide (VIP) supplying the cerebral arteries were studied with immunohistochemistry in rats. The innervation patterns and densities of NPY and VIP fibers were similar at all stages of development and similar to that previously reported for norepinephrine (NE). There was a striking reorganization of the innervation pattern of all three fiber systems between the first and second postnatal weeks. At all stages of development prior to the first postnatal week, growth cones were present on individual fibers at the distal part of major cerebral arteries and the middle segment of the basilar artery. The growth cones had a range of shapes from blunt to stellate, lanceolate or filiform. NPY and VIP immunoreactive granules were commonly present. The present results taken with our earlier developmental study of NE fibers (J. Comp. Neurol., 271 (1988) 435-444), demonstrate that: (1) both sympathetic and parasympathetic perivascular nerves on immature cerebral vessels develop with similar sequences: first longitudinal fibers and fiber bundles are present; these transform to a meshwork pattern and finally transform again into the mature, predominantly circumferential pattern; (2) both the classical (NE) and peptidergic transmitters (NPY) within the sympathetic system appear to develop identically in terms of time of appearance, innervation patterns, densities and reorganization.(ABSTRACT TRUNCATED AT 250 WORDS)


Interendothelial junctions during blood-brain barrier development in the rat: morphological changes at the level of individual tight junctional contacts. The endothelium of brain capillaries represents the structural basis for the blood-brain barrier in vertebrates. Individual endothelial cells are linked by a continuous belt of complex tight junctions (zonulae occludentes). Hydrophilic solutes and macromolecules are believed to cross the barrier through specific carrier mechanisms. Unspecific paracellular ionic leak is thought to be very low. In rats the blood-brain barrier is not fully developed until postnatal day 24. We investigated the ultrastructure of the developing blood-brain barrier at 5 developmental stages between embryonic day 17 and young adults. The use of high power goniometric tilting of ultrathin sections allows one to gather information about the exact relationship between two opposing membranes throughout the entire length of the cleft. Our results suggest that the maturation of blood--brain barrier interendothelial clefts is accompanied by the establishment of a characteristic ratio of 'narrow zone' (complex tight junctions) to 'wide zone' (15-20 nm), and of a typical cleft length. Membrane separation larger than 20 nm disappear and individual tight junctional contacts undergo structural changes.


Axotomized frog sciatic nerve releases diffusible neurite-promoting factors. Using the bullfrog (Rana catesbeiana) dorsal root ganglia (DRG) and its sciatic nerve (ScN) as a model system, we have previously described neuronal and non-neuronal molecular changes associated with the early regenerative response of DRG neurons to axotomy. Since diffusible molecular factors, released by axotomized ScN, might function to stimulate axon regrowth, we have assayed the ability of ScN-conditioned bath to promote in vitro neurite outgrowth from PC-12 cells. Diffusible ScN proteins were collected by incubating segments of normal or axotomized ScN in a small volume of RPMI media for 4 h (nerve bath). The nerve baths, supplemented with serum, were then added to PC-12 cell cultures to assay for the presence of neurite growth factors released by ScN. Results showed that nerve baths, collected from sham-operated or axotomized ScN, could not induce the differentiation of PC-12 into neurite-bearing cells. Therefore, in all subsequent neurite growth assay experiments, an exogenous source of nerve growth factor (NGF) (50 ng/ml) was added to the nerve baths or unconditioned media to generate and maintain PC-12 neuritic structure. We found that nerve baths, collected from previously axotomized (at least 3 days post-injury) nerve, contained diffusible factors which enhanced PC-12 neurite growth, relative to unconditioned media. No neurite growth factors were observed to be released by sham-operated ScN or 1-day post-axotomized ScN. Further experiments were conducted to identify the diffusible neurite growth factors released from axotomized ScN. We showed that the release (if any) of endogenous diffusible NGF or laminin from axotomized nerve could not have accounted for the facilitation of neurite growth. Analysis of radiolabelled ScN proteins by two-dimensional polyacrylamide gel could not have accounted for the facilitation of neurite growth. Analysis of radiolabelled ScN proteins by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) showed that the relative abundance of two diffusible proteins (M(r) approximately 35 and 70 kDa) in the nerve bath was directly correlated with the ability of the nerve bath to facilitate PC-12 neurite growth.


A PCR analysis of rhodopsin gene transcription in rat pineal photoreceptor differentiation. A polymerase chain reaction (PCR) method was used to detect rhodopsin transcripts in rat pineals both in vivo and in vitro. Only very low levels of transcripts were detected in preparations from adult rat pineal tissue, but fairly large amounts were detected in cDNA preparations from cultures of newborn rat pineals. The transcript level was reduced significantly if the cells had been cultured in the presence of 10 microM norepinephrine (NE). This concentration of NE had previously been shown to abolish almost completely the rhodopsin immunoreactivity normally seen in such cultures. The present work indicates that part, but not all, of the effect of NE is probably at the level of translation.


Thyrotropin releasing hormone (TRH) and its analog, RGH-2202, accelerate maturation of cerebellar neurons in vitro. We have studied the "trophic" action of thyrotropin releasing hormone (TRH) in cultured cerebellar granule cells, a pure and homogeneous population of glutamatergic neurons. As an index of neuronal maturation, we have measured the uptake of D-[3H]aspartate (a non-metabolizable analog of glutamate) at different days of maturation in vitro (DIV). In control cultures, D-[3H]Aspartate increased linearly during maturation reaching plateau values between 7 and 9 DIV; daily addition of TRH tartrate (TRH-t) or RGH-2202 (a TRH analog) accelerated in a concentration-dependent manner the maturation profile of D-[3H]aspartate uptake. This effect was more pronounced for RGH-2202: in cultures treated daily with RGH-2202, D-[3H]aspartate uptake was fully expressed after 3 DIV. Neither TRH-t nor RGH-2202 significantly increased D-[3H]aspartate uptake in mature cells, excluding a direct action on the glutamate transport system. Both compounds specifically potentiated the increase in [3H]inositol monophosphate formation (but not the stimulation of 45Ca2+ influx) induced by N-methyl-D-aspartate (NMDA) receptor agonists, without affecting the stimulation of inositol phospholipid hydrolysis by quisqualate or carbamylcholine. We suggest that, in cultured cerebellar granule cells, TRH and RGH-2202 enhance the trophic action of endogenous glutamate by amplifying some of the intracellular events that follow the influx of extracellular Ca2+ through NMDA-gated ion channels.


Sex differences in cortical thickness and the dendritic tree in the monocular and binocular subfields of the rat visual cortex at weaning age. The visual cortex of adult rats is sexually dimorphic at both the gross size and dendritic levels [Brain Res., 295 (1984) 27-34; J. Comp. Neurol., in press]. In addition, sex differences in the dendritic tree are dynamic and can be altered by environmental conditions imposed at weaning [Brain Res., 295 (1984) 27-34]. The present study examines sex differences in cortical thickness and in the dendritic tree of the monocular (Oc1M) and binocular (Oc1B) subfields in littermate male and female pairs of Long-Evans rats at weaning age (25 days). From Nissl-stained tissue, it was found that the whole cortex and layer II-IV of Oc1B was thicker in males than females. No sex differences were found in the thickness of Oc1M. Golgi-Cox-stained pyramidal neurons in layer III from the Oc1M and Oc1B regions were quantified in 7 littermate pairs of weaning-age rats. There were no sex differences in the basilar tree, while the apical oblique branches were sexually dimorphic, especially in the monocular region. Females had greater total dendritic length and longer terminal branches in Oc1M compared to males. Females also had longer bifurcating branches in both Oc1M and Oc1B than males. The present study found that sex differences at weaning age do not completely mirror the dimorphisms found in the visual cortex of the adult rat. This study also indicates that related subfields can differ in their morphology and should be examined separately.


Laminin selectively enhances axonal growth and accelerates the development of polarity by hippocampal neurons in culture. We have examined the effects of laminin on the morphological development of embryonic rat hippocampal neurons maintained in tissue culture. Forty-eight hours after plating, neurons grown on a polylysine-coated substrate had become polarized, typically having one long axon and 4 or 5 minor processes. Adsorption of laminin to the substrate did not cause changes in the number of axons extended by hippocampal neurons but did cause significant increases in the length of the axonal plexus and in axonal branching. In contrast to its effects on axons, laminin did not influence the number, length, or branching of the minor processes that eventually become dendrites or the morphology of definite dendrites as assessed after 7 days in culture. In addition to selectively enhancing axonal growth, laminin greatly increased the rate of polarization of hippocampal neurons such that most became polarized within 18 h. Analysis of the time course of laminin's effects revealed that the acceleration of polarization was not associated with a change in the time of initial process formation, but rather with a selective stimulation of the growth of the longest process at all times from the 12th through the 48th h in vitro. These data suggest that even though the basic shape of hippocampal neurons may be intrinsically programmed, critical aspects of their morphological development may be modulated by extracellular matrix molecules such as laminin.


Alterations in septohippocampal cholinergic innervations and related behaviors after early exposure to heroin and phencyclidine. Mice were exposed to diacetylmorphine (heroin) or phencyclidine (PCP) prenatally or neonatally. At a later age, they were tested for hippocampus-related behavioral deficits and concomitant alterations in the septohippocampal cholinergic innervations. Actually, this is an application of the previously established phenobarbital neuroteratogenicity model to heroin and PCP. Prenatal exposure was accomplished transplacentally by injecting the mother 10 mg/kg heroin or PCP on gestation days 9-18. Neonatal administrations were applied directly by injections of 10 mg/kg of either drug to the pups between neonatal days 2-21. At the age of 50 days, mice exposed to heroin and PCP prenatally exhibited a 107% and 159% increase in their muscarinic cholinergic receptors Bmax, respectively. Neonatal exposure to heroin or PCP caused an 83% and 76% increase in the receptors respectively. On the behavioral level, both prenatal and neonatal exposure to heroin or PCP reduced performance in the hippocampus related eight-arm maze and Morris mazes. Depending on the drug, the test and the period of drug administration, the reduction ranged between 10% and 75%. The results suggest that heroin and PCP induce alterations in the septohippocampal cholinergic innervations and in related behavioral performance. Further studies are necessary in order to connect the biochemical and behavioral events in causal relationships.


Characterization of a novel set of membrane antigens associated with axonal growth. II. Expression in the chick central nervous system. In the preceding paper (Dev. Brain Res., 69 (1992) 215-223), a polyclonal antiserum ('3070') and several monoclonal antibodies (MAbs 4 and 199) were used to define a novel set of laminin-binding membrane antigens in chick brain. These MAbs specifically inhibited attachment of NG108-15 cells to low, but not high amounts of laminin. Here, the immunocytochemical localization of MAb 4 and 3070 antigens was examined in frozen sections of E3-P21 chick brain, with particular reference to retina, optic tectum, cerebellum and spinal cord. Growing axons and migrating nascent neurons were stained transiently, while projection neurons were intensely labelled at all stages. Some interneurons and glial cells were stained faintly; others, apparently not at all. Finally, 3070 antiserum stained neuropil regions in mature brain. Cells in the CNS expressing high levels of MAb 4 antigens corresponded to those which, in rodents, are known to migrate in response to laminin or to synthesize forms of laminin themselves. These data, together with evidence presented elsewhere (J. Comp. Neurol., 313 (1991) 625-642) and in the next paper in this series (Dev. Brain Res., 69 (1992) 277-282), suggest that MAb 4 and 3070 antigens are important in neural development by mediating at least some of the responses of neural cells to laminin--for example, by acting as a laminin receptor guiding axonal outgrowth and neuronal migration, or by involvement in the transport and binding of laminin to the surface of neurons and reactive glial cells.


An experimental model of generalized seizures for the measurement of local cerebral glucose utilization in the immature rat. I. Behavioral characterization and determination of lumped constant. An experimental model of status epilepticus has been developed in the immature rat by administration of pentylenetetrazol (PTZ) using repetitive, timed intraperitoneal injections of subconvulsive doses. The pattern of behavioral signs has been well characterized in each age group, i.e. 10 (P10), 14 (P14), 17 (P17) and 21 postnatal days (P21). In this model, the dose of convulsant could be adjusted as a function of interindividual sensitivity and status epilepticus lated for quite a long duration to allow the measurement of local cerebral metabolic rates for glucose (LCMRglc) by means of the [14C]2-deoxyglucose method [J. Neurochem., 28 (1977) 897-916]. To estimate LCMRglc during status epilepticus, the lumped constant (LC) was re-calculated in controls and PTZ-treated rats. The control LC was 0.54 at P10 and 0.50-0.51 at the three older ages studied (P14, P17 and P21). During status epilepticus, it increased to 0.64 in P10 rats and decreased to 0.42 and 0.40, respectively, in P17 and P21 animals. At P14, LC was not affected by seizures. The measurements of brain lactate levels showed a large 4.5-10-fold increase in PTZ-treated rats as compared to controls at all ages. The results of the present study show that the immature brain responds to sustained seizure activity in a specific way according to its postnatal age. Moreover, our results underscore the necessity of re-calculation of LC to the quantification of LCMRglc in such pathological states, particularly in immature animals.


An experimental model of generalized seizures for the measurement of local cerebral glucose utilization in the immature rat. II. Mapping of brain metabolism using the quantitative [14C]2-deoxyglucose technique. The quantitative autoradiographic [14C]2-deoxyglucose technique (2DG) was applied to measure the effects of pentylenetetrazol (PTZ)-induced status epilepticus (SE) on local cerebral metabolic rates for glucose (LCMRglc) in 10 (P10)-, 14 (P14)-, 17 (P17)- and 21 (P21)-day-old rats. To produce long-lasting SE (55 min), the animals received repetitive, timed intraperitoneal injections of subconvulsive doses of PTZ until SE was reached. At P10 and P14, SE induced a marked increase in LCMRglc which affected 66 of the 76 structures studied. Increases were especially high (200-400%) in limbic and motor cortices at P10 and in some brainstem areas at these 2 ages. At P17 and P21, average brain glucose utilization was similar in seizing and control rats, but in PTZ-treated rats reflected a redistribution in local metabolic rates with increases in brainstem, midbrain, hypothalamus and septum, decreases in cortex, hippocampus, some sensory areas and white matter and no change in many motor and limbic structures. In a few cerebral regions, such as hippocampus, dentate gyrus and mammillary body, LCMRglc did not increase at P10 and P14 and decreased at P17 and P21 in PTZ- vs. saline-treated rats. The results of the present study show that the immature brain responds to sustained seizure activity in a specific way according to its maturational state. Moreover, these data allow the mapping of the vulnerability of cerebral structures to seizures, according to their metabolic response to convulsions.


Distribution and relative density of p75 nerve growth factor receptors in the rat spinal cord as a function of age and treatment with antibodies to nerve growth factor. It has been postulated that nerve growth factor (NGF) binding to the low-affinity fast-dissociating NGF receptor (p75 NGFR) on Schwann cells and growing neurites is involved with the molecular feedback necessary for continued neurite extension during development and regeneration. Since central projections of somatosensory fibers sprout into the spinal cord after daily neonatal injections of antibodies to NGF (ANTI-NGF) for a one month period, it is of interest to determine if the distribution of p75 NGFR correlates with the occurrence of sprouting. Spinal cords from three groups of rats: untreated, preimmune sera treated and ANTI-NGF treated were examined on postnatal days (PD) 0, 14 and 30. The p75 NGFR distribution was determined using the monoclonal antibody 192 with standard immunohistochemical techniques and the optical density of the immunoreaction product was quantified using an Amersham image analysis system. The 192 immunoreaction product was localized to laminae I-IV, the dorsal columns, the dorsolateral funiculus, Lissauer's tract (LT) and the ventral horn on PD 0; to laminae I-III and medial IV and LT on PD 14; and laminae I-II and LT on PD 30. The untreated and preimmune sera treated groups show no difference in distribution. In the ANTI-NGF treated group, the 192 immunoreaction product was localized to laminae I-V and LT on PD 14 and to laminae I-III and medial IV and LT on PD 30. Similarly, the optical density of the ANTI-NGF treated group was significantly greater than same aged untreated and preimmune sera treated groups, but was not statistically different from these two groups examined 14 days earlier. Thus, ANTI-NGF treatment interferes with the postnatal downregulation of p75 NGFR in the dorsal horn and may provide for continued neurite growth.


Effects of the subcutaneous administration of enkephalins on tail-flick response and righting reflex of developing rats. The s.c. administration of [Met5]-enkephalin to 10-day-old rats pretreated with the mixture of 3 peptidase inhibitors, amastatin, captopril and phosphoramidon, produced the inhibition of tail-flick response and loss of righting reflex. When infant rats were pretreated with the mixture of any combination of two peptidase inhibitors, however, the change in both the response and the reflex were not produced at all by enkephalin injection, indicating that 3 kinds of enzymes, amastatin-sensitive aminopeptidase(s), captopril-sensitive peptidyl dipeptidase A and phosphoramidon-sensitive endopeptidase 24.11, played an important role in the inactivation of enkephalin after its systemic administration. Additionally, the fact that the two enkephalin-induced effects were more effectively antagonized by naloxone, a relatively selective mu-opioid antagonist, than by naltrindole, a specific delta-antagonist, or by nor-binaltorphimine, a specific kappa-antagonist, showed that these two effects were produced by the interaction of enkephalin with mu receptors. Moreover the involvement of mu receptors in the production of these two effects was shown by the fact that the s.c. administration of [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, a selective mu agonist, also produced these two effects which were more effectively antagonized by naloxone than by naltrindole or nor-binaltorphimine. Since the magnitude of the two effects induced by enkephalins in 15-day-old rats was significantly lower than that in 10-day-old rats, and the two enkephalin-induced effects were not produced at all in 20-day-old rats, a maturation-induced decrease in the permeability of the blood-brain barrier against opioid peptides was indicated.


Characterization of novel set of membrane antigens associated with axonal growth. III: Expression in the regenerating goldfish optic nerve and tectum. In the preceding two papers in this series, a polyclonal antiserum (3070) and several monoclonal antibodies (MAbs 4 and 199) were used to define a novel set of laminin-binding membrane antigens in chick brain. In chick brain, 3070 and MAb 4 antigens stained growing axons and migrating nascent neurons transiently; though projection neurons were intensely labelled at all stages, normal glial cells were stained faintly or not at all. To learn if these antigens are associated more generally with active states of nerve growth, the present paper examined the expression of 3070 and MAb 4 antigens during regeneration of the goldfish optic nerve. Only a few optic fibers or glial cells were stained by 3070 antiserum in the resting (uninjured) optic nerve, but 10 days following a unilateral nerve crush, when the growth response is maximal in these fish, distal regenerating neuritic sprouts and glial cell bodies and processes were profusely labelled. The intensity of labelling was diminished by 135 days post-crush, when most active growth was completed. Since 3070/MAb 4 antigens exhibit properties expected for functional laminin receptors, these findings suggest several possible roles that they could play to aid nerve regeneration.


Fetal nicotine exposure ablates the ability of postnatal nicotine challenge to release norepinephrine from rat brain regions. Exposure of the fetus to nicotine is known to affect the function of noradrenergic pathways in the central nervous system. In the current study, synaptic mechanisms underlying the functional defects were evaluated in the offspring of pregnant rats given nicotine infusions of 2 mg/kg/day throughout gestation, administered by osmotic minipumps. At 30 days postpartum, norepinephrine levels in brain regions of the offspring were significantly reduced. More importantly, acute challenge with either 0.1 mg/kg or 0.3 mg/kg of nicotine evoked significant norepinephrine release from brain regions of control animals, but failed to do so in the fetal nicotine cohort. These results suggest that prenatal exposure to nicotine produces a deficit in subsequent noradrenergic responsiveness, deficits which may participate in behavioral and neuroendocrine abnormalities.


Parathyroid hormone-related protein: structure, function, and measurement. Parathyroid hormone-related protein (PTHRP) is a 139- to 173-amino-acid protein with N-terminal homology to parathyroid hormone (PTH). Initially isolated from tumors from patients with humoral hypercalcemia of malignancy, PTHRP appears to be of far more widespread physiological importance. Its complex gene is expressed in a surprising diversity of tissues. The primary amino acid sequences of both the PTH-like and non-PTH-like regions of the protein are highly conserved across species. In addition to classical and nonclassical PTH-like activities of the N-terminal region, other biological functions have been ascribed, including augmentation of calcium transport by midregion PTHRP and potent inhibition of bone resorption by a C-terminal peptide. There is emerging evidence that the protein undergoes extensive processing, including glycosylation and defined proteolytic cleavages. Several region-specific immunoassays are now capable of measuring circulating concentrations of PTHRP in patients with humoral hypercalcemia of malignancy. Molar concentrations of different regions may differ by more than an order of magnitude, reflecting diversity in processing and (or) metabolism. By analyzing the results of these assays, taking into account the specificities of known endoproteases, one can hypothesize about some of the endoproteolytic cleavages that occur in PTHRP metabolism. Clinically, selected PTHRP assays can be very helpful in diagnosing PTHRP-mediated hypercalcemia, but are not yet sufficiently sensitive to accurately measure normal concentrations of the protein.


Washing erythrocytes to remove interferents in measurements of zinc protoporphyrin by front-face hematofluorometry. Zinc protoporphyrin (ZPP) is determined by hematofluorometry of whole blood to detect iron deficiency in blood donors. In hospitalized patients, ZPP did not correlate with established markers of iron status. We performed 4500 ZPP measurements with the Aviv front-face hematofluorometer in samples from 475 patients and measured ferritin, transferrin saturation, hemoglobin, and erythrocyte indices. We found that the fluorometric determination is affected by substances dissolved in plasma but that this interference can be eliminated by using washed erythrocytes. In validation tests the within-day variation was < 3.5%; the day-to-day variation was < 6.8%. In 130 healthy persons without iron deficiency, ZPP was < or = 40 mumol/mol heme, which we consider a normal value. Mean ZPP in 46 iron-deficient patients was 256 (SD 105) mumol/mol heme (correlation with ferritin: -0.73; with hemoglobin: -0.85; P < 0.001). When washed erythrocytes are used, the hematofluorometric determination of ZPP is sensitive and specific for detecting iron deficiency in otherwise healthy individuals and hospitalized patients.


Influence of obesity on plasma fructosamine concentration. The influence of obesity on plasma fructosamine concentration was studied in 68 diabetic and 1335 nondiabetic subjects from a Chinese community. Obese nondiabetic men (body mass index > 25 kg/m2) had lower fructosamine concentrations than nonobese nondiabetic men (body mass index < or = 25 kg/m2); the pattern was similar for diabetic women. Stepwise multiple-regression analysis showed that, apart from known factors (total protein, albumin, and indices of glycemic control), fructosamine was also associated with body mass index and plasma fasting triglycerides. However, the contribution of these were small except in diabetic women. We conclude that the effect of obesity on fructosamine is small.


Specific assay of serum lactate dehydrogenase isoenzyme 1 by proteolysis with alpha-chymotrypsin and protein denaturation. We devised a method for assaying serum lactate dehydrogenase isoenzyme 1 (LD-1) activity specifically by preincubation with alpha-chymotrypsin and guanidine. Cleavage of phenylalanine bonds in the loop of A and B subunits of LD-3, LD-4, and LD-5 isoenzymes (residues 117-119) by incubation with alpha-chymotrypsin for a short time completely inactivated these isoenzymes and partially inactivated LD-2. Addition of guanidine (0.50 mol/L, pH 7.8) to the incubation mixture containing the chymotrypsin completed the inactivation of LD-2. As much as 4000 U/L of LD-2, LD-3, LD-4, and LD-5 were inactivated, whereas LD-1 was affected only slightly. Results by this method (y) correlated well with those by the Roche Isomune immunochemical LD-1 method (x): y = 0.98 x -0.11, r = 0.99 (n = 60). Within-run CVs were 0.5-2.5%. Several common interferents had no effect. In 500 healthy people, serum LD-1 ranged between 66 and 130 U/L, with a mean +/- SD of 88 +/- 15 U/L.


Using 16O35Cl to correct for chloride interference improves accuracy of urine arsenic determinations by inductively coupled plasma mass spectrometry. We have observed inaccurate urine arsenic values with the method of isobaric fractionation, which was designed to correct for the 40Ar35Cl interference with 75As quantitation by inductively coupled plasma mass spectrometry. Isobaric fractionation, which is based on ion intensities at m/z 77 and 82, consistently underestimates the 40Ar35Cl interference and overestimates urine arsenic. We present an improved method for identifying the argon-chloride interference. We observed that signal intensities for the species 16O35Cl and 40Ar35Cl are proportional (I75 = 0.0295 x I51 - 14.7, r2 = 0.998; where Ix is the normalized ion intensity at m/z X) in water and urine, over a broad range of chloride concentrations (0-800 mmol/L). The proportionality constant is remarkably stable within a run (mean and SD, 0.0295 +/- 0.0023, based on 10 replicates of five chloride calibrators, 0, 100, 200, 400, and 800 mmol/L). Increased sensitivity (50-fold) for detecting the 40Ar35Cl interference provides improved accuracy for urine arsenic quantitation as demonstrated by a split-sample comparison with graphite-furnace atomic absorption spectrophotometry.


Development of monoclonal antibodies for an assay of cardiac troponin-I and preliminary results in suspected cases of myocardial infarction. To improve the specificity of biochemical markers of myocardial infarction (MI), we have developed a double monoclonal "sandwich" enzyme immunoassay to measure cardiac troponin-I (cTnI) in serum. We produced eight IgG monoclonal antibodies against human cardiac troponin-I (cTnI) and tested them against human and animal (canine, bovine, and rabbit) troponins. Five antibodies were cardiac-specific; none of the antibodies were species-specific. Two of the five cTnI-specific monoclonal antibodies were utilized in an immunoassay. Standards were made by adding purified human cTnI to affinity-stripped cTnI-free human sera to cover the range 0-100 micrograms/L for cTnI. The dose-response curve was nonlinear but reproducible. Total assay imprecision (CV) varied between 11% and 21%. The upper limit of the reference range (nonparametric 95% interval) was established as 3.1 micrograms/L by measuring cTnI concentration in sera of 159 hospitalized patients without evidence of cardiac disease. Purified human skeletal TnI up to 10,000 micrograms/L did not affect the assay (calculated cross-reactivity < 0.1%). Diagnostic sensitivities of creatine kinase MB isoenzyme (CK-MB) and cTnI were evaluated retrospectively in 49 consecutive patients with proven MI. In the 30 patients for whom sufficient information was available to establish an accurate time course, CK-MB was more sensitive during the first 4 h after the onset of chest pain, but thereafter the sensitivities were similar up to 48 h. However, cTnI is more cardiac-specific than is CK-MB and remains increased longer than does CK-MB.


Automated analysis for free and short-chain acylcarnitine in plasma with a centrifugal analyzer. We describe a fully automated, spectrophotometric assay of free and total carnitine in plasma ultrafiltrates. The method, suitable for routine application in most hospital laboratories, incorporates the hydrolysis of acylcarnitines to free carnitine within the program of a Cobas Fara II centrifugal analyzer. The hydrolysis is monitored and calibrated with standard solutions containing octanoylcarnitine. Results correlated well with those from a reference isotope-dilution mass spectrometric assay. The ability to analyze a batch of samples for both free and total carnitine within 90 min enables analysis of > or = 100 samples per day. Used in conjunction with acylcarnitine species identification by mass spectrometry, the Cobas assay facilitates the diagnosis of carnitine-deficiency syndromes and specific metabolic disorders.


Simple method for correcting total protein in plasma for actual fibrinogen content. Using plasma instead of serum for routine chemistry analyses has many advantages. To overcome the disadvantage of inclusively measuring fibrinogen in the plasma total protein assay without changing the clinical significance of the total protein assay, we investigated the possibility of subtracting the actual amount of fibrinogen from the plasma total protein. The correlation between serum and plasma total protein was excellent (plasma total protein = 0.989 x serum total protein + 6.7 g/L; r = 0.969; n = 131; mean difference = 5.55 g/L; P < 0.001). When the plasma total protein was corrected for the actual amount of fibrinogen, the correlation with serum total protein was equally good but the intercept was practically eliminated (corrected plasma total protein = 1.009 x serum total protein + 0.25 g/L; r = 0.985; n = 131; mean difference = 0.78 g/L; P = 0.47). The mean concentration of fibrinogen was 2.5 g/L (range: 1.38-3.62 g/L; n = 404) for blood donors, 3.6 g/L (n = 2707) for patients from the outpatient department, 4.6 g/L (n = 2023) for patients admitted to the hospital, and 6.6 g/L (n = 219) for patients whose concentration of C-reactive protein was > 50 mg/L. We conclude that the plasma total protein result should be corrected for the actual amount of fibrinogen.


Ratio of creatine kinase 2 mass concentration to total creatine kinase activity not altered by heavy physical exercise. Serum creatine kinase isoenzyme 2 concentrations (CK 2 mass) were measured in marathon runners during training and 1 and 2 days after a race and compared with values from 36 acute myocardial infarction (AMI) patients whose total CK and (or) CK 2 activities were similar to those of runners in the basal state. During training, runners had CK and CK 2 activities 53% and 43% above reference values, respectively, and 36% had CK 2 activity > 5% of total CK. Nine runners (26%) showed CK 2 mass values > 6 micrograms/L but < or = 10 micrograms/L; 35 of the AMI subjects, despite having CK activities similar to those of runners, had values > 10 micrograms/L. The ratio of CK 2 mass to total CK activity was significantly (P < 0.0002) different between sexes for runners. At 1 and 2 days after racing, 100% of CK and CK 2 activities and 71% and 57% of the percentages of CK 2 activity, respectively, were abnormally high; 57% and 43% of CK 2 mass values were > 10 micrograms/L, being comparable with those observed for the AMI group. Basal CK 2 mass values of the runners appeared only slightly higher than that for sedentary subjects, but after exercise half the subjects presented increased values similar to those observed for AMI subjects. The ratio of CK 2 mass to total CK activity appeared unaltered by exercise in all but one of the samples assayed, indicating its utility in evaluating CK 2 mass increases originating in skeletal muscle.


Cross-reactivity of anti-digoxin antibodies with digitoxin depends on tracer structure. The most common methods for measuring digoxin concentrations in serum are immunoassays. The prerequisite for exact determination of the digoxin value is an antibody that specifically binds digoxin. Because digitoxin differs from digoxin only in the C-12 hydroxy group, it is difficult to obtain anti-digoxin antibodies that do not cross-react with this compound. During the development of a fluorescence polarization immunoassay (FPIA) for digoxin, we investigated digoxin tracers with different structures. We found that in FPIA the digitoxin cross-reactivity of an antibody could be reduced by varying the structure of the tracer molecule.


Sandwich immunoassay for intact human osteocalcin. To overcome the problems of limited-region specificity associated with conventional radioimmunoassay (RIA), we developed a sandwich enzyme immunoassay (EIA) for intact human osteocalcin (hOC). For this EIA we used antibodies to the N- and C-terminal regions of hOC that were raised against an N-terminal 20-residue peptide and a C-terminal 7-residue peptide, both synthetic. Immunoassay profiles of tryptic digests of hOC and serum from patients with renal failure, fractionated by reversed-phase HPLC, facilitated direct demonstration of the region specificity of this method. A preliminary study of serum osteocalcin concentrations in patients with renal failure further confirmed this specificity, showing lower positive rates obtained by this method than by conventional RIA. The cross-reactivity data of hOC with bovine and rat osteocalcins by the sandwich method indicated its species specificity. These studies demonstrate the superior specificity of this sandwich EIA compared with conventional RIA and thus confirm its potential diagnostic superiority.


Apolipoprotein(a) is present in urine and its excretion is decreased in patients with renal failure. To investigate the relation between renal function and concentrations of lipoprotein(a) [Lp(a)] in serum, we measured Lp(a) in samples of serum and urine from patients with diabetes mellitus and in samples sent to a laboratory center for measurements of creatinine clearance. Serum Lp(a) concentrations were significantly increased in subjects with obvious renal dysfunction (serum creatinine > or = 176.8 mumol/L) compared with normal control subjects. Urinary Lp(a) excretion was decreased in subjects with obvious renal dysfunction compared with subjects without obvious renal dysfunction (serum creatinine < or = 88.4 mumol/L) and was negatively and positively correlated with serum creatinine and creatinine clearance, respectively. More than 80% of urinary Lp(a) was recovered in the d > 1.21 kg/L fraction. At least six bands for apolipoprotein(a) [apo(a)] fragments, which were smaller than native apo(a) in serum, were observed in urine by immunoblotting, and some of these were also detected in serum. Degraded apo(a) fragments are probably present in urine, and their excretion decreases in parallel with decreases in the glomerular filtration rate.


Rapid nonradioactive tracer method for detecting carriers of the major Ashkenazi Jewish Tay-Sachs disease mutations. Tay-Sachs disease (TSD, GM2 gangliosidosis, Type I) is an autosomal recessive lysosomal storage disease caused by deficiency of beta-hexosaminidase A (Hex A) resulting from mutations in the gene (HEXA) encoding the alpha-subunit of the enzyme. Three mutations, in exons 7 and 11 and at the exon 12-intron 12 junction, account for > 90% of alleles identified in obligate Ashkenazi Jewish carriers. Mutation analysis requires amplification of available DNA by separate polymerase chain reactions (PCRs) and either restriction digestion and gel electrophoresis or 32P-labeled allele-specific oligonucleotide (ASO) probes. We developed a simple, nonradioisotopic method for rapidly identifying TSD carriers by a triplex PCR reaction followed by dot-blot analysis, using three wild-type and three mutant ASOs end-labeled with digoxigenin-dUTP (dig-ASO). Hybridization was demonstrated immunologically by reaction with an anti-digoxigenin-alkaline phosphatase conjugate followed by colorimetric demonstration of phosphatase activity. The results of analyses by the dig-ASO method of 65 carriers identified by serum enzyme activity and of 6 high-risk fetuses in prenatal testing were the same as those obtained by more conventional restriction analysis. Dig-ASO testing correctly reclassified 10 individuals who had tested inconclusively on analysis for leukocyte beta-hexosaminidase A activity; 3 were identified as carriers and 7 as noncarriers. The simplicity of the assay and the avoidance of the radioisotopes make this a potentially useful method for TSD carrier detection by mutation analysis in Ashkenazi Jews from populations in whom the identity and frequencies of the common TSD mutations are known.


Analytical goal-setting for monitoring patients when two analytical methods are used. Serial results from an individual are often obtained using more than one method. Results should be transferable over time and locale. Every method has inherent analytical error, and goals are required to delineate the maximum allowable random (imprecision) and systematic (inaccuracy, bias) errors to facilitate optimal patient care. Based on Harris's proposal [Am J Clin Pathol 1979;72:374-82] that desirable imprecision should be less than or equal to one-half the within-subject biological variation, if the methods have negligible imprecision, then the maximum allowable bias between two methods used for monitoring is one-third of the within-subject biological variation. A more general model has been developed that relates the analytical imprecisions of two methods, and the bias between them, to biological variation. Applying the general formula derived in specific clinical monitoring situations in which a known change in serial results (occurring at a stated probability) stimulates clinical action allows goals for the imprecisions of the two methods and allows the difference in bias between them to be determined quantitatively.


Plasma lipoprotein(a) values and severity of coronary artery disease in a large population of patients undergoing coronary angiography. To determine possible associations between lipoprotein(a) [Lp(a)] and the severity of coronary artery lesions, we measured lipid, apolipoprotein, and Lp(a) in a large population of Belgian patients (n = 1054) undergoing coronary angiography. In both women and men, univariate analysis demonstrated significant differences in the Lp(a) concentrations according to the severity of the coronary stenosis. However, after adjustment for possible confounding factors, many of these differences were attenuated, indicating that other variables that differentiate patients from control subjects also influence Lp(a) distribution. Differences in lipid, apolipoprotein, and Lp(a) concentrations between male and female patients are discussed.


Immunoluminometric assay of chromogranin A in serum with commercially available reagents. Chromogranin A (Cg A) is a useful marker of neuroendocrine neoplasia in humans. Here we describe an immunoluminometric assay (ILMA) for measuring Cg A in serum, with use of a new tube as a solid phase. The new tube has a large surface area and is coated with a polyclonal antibody. Optimized coating conditions provide a high IgG adsorption to the polystyrene wall. Serum is added to the coated tube and incubated for 2 h or overnight. After washing the tubes, acridinium ester-labeled monoclonal antibody against Cg A is added and incubation is continued for 2 h. The tubes are washed again and the bound luminescence is measured. The assay is very sensitive (detection limit 1 microgram/L, if 25 microL of serum is used), is specific for human Cg A, and offers a wide dynamic range (1-1000 micrograms/L). The range in healthy humans is 10-53 micrograms/L (median 30 micrograms/L). The correlation of serum Cg A to creatinine and parathyroid hormone is described. We also report the influence of intravenous calcium injection on Cg A concentrations in serum.


Pre-beta high-density lipoprotein determined by immunoblotting with chemiluminescent detection. We describe a novel assay of pre-beta high-density lipoprotein (HDL), a unique apolipoprotein A-I (apo A-I)-containing lipoprotein particle. The pre-beta and alpha lipoproteins are separated by electrophoresis in agarose and transferred onto a membrane by capillary blotting. The membrane blot is sequentially incubated with sheep anti-human apo A-I antiserum and then with a conjugate of rabbit anti-sheep immunoglobulin and horseradish peroxidase. Chemiluminescence formed by the peroxidase-catalyzed oxidation of luminol in the presence of an enhancer is captured on photographic film, and the pre-beta HDL band is quantified by transmission densitometry. The assay is calibrated with standards prepared from a reference serum diluted in 9 mol/L urea. Within-batch precision (CV) at pre-beta HDL concentrations of 22.1 and 44.3 mg/L was 7% and 4.9% respectively. Pre-beta HDL contained 1.6% (0.65-2.6%, mean and range) of total serum apo A-I in 30 normolipidemic subjects.


Pitfalls in the measurement of plasma osmolality pertinent to research in vasopressin and water metabolism. The reliability of measurements of plasma osmolality is known to be biased by technical artifacts, such as the anticoagulant and the osmometric technique used; the resulting measurement errors therefore may cause errors in interpretation of data. In assessing the potential biasing influence of procedural variables, we found that the temperature at which fresh plasma samples were stored, the duration of storage, and the freezing and thawing of samples appeared to significantly (P < 0.01) affect osmolality values around the narrow physiological range. These factors should be considered in the interpretation of studies on the osmoregulation of vasopressin secretion. In particular, the results suggest that data obtained for any but fresh samples, whether frozen-thawed samples or samples stored at room temperature, are unreliable.


Abbott IMx and serono MAIAclone assays compared for lutropin determinations in urine. The urinary lutropin (luteinizing hormone; LH) profiles of 20 patients were determined in 10 consecutive urine samples (n = 200) with a manual technique (MAIAclone, Serono; y) and a fully automated (Abbott IMx; x) technique. For 132 single determinations within the range of detection, the regression line was y = 1.39 x - 0.07 (r = 0.85). Retrospective analysis of the LH profiles obtained by the IMx yielded the classification "not pregnant" (negative) in 14 cases, of which 13 were correctly classified; 6 profiles were classified as "pregnant" (positive), which correlated with the clinical findings in 4 cases. Nine of the LH profiles analyzed with the MAIAclone method were classified as negative, which was correct in seven cases. Retrospective analysis of 11 LH profiles established with the MAIAclone assay gave positive results, which agreed with the clinical outcome in three cases. The diagnostic sensitivity of the IMx and MAIAclone methods was 87% and 47%, respectively; diagnostic specificity was 80% and 60%, respectively. In smaller series (< or = 40 samples), the IMx method has considerable advantages over the MAIAclone method in cost and convenience, because standard curves can be stored and assays can be performed as single determinations.


Investigation of N-methyl-D-glucamine buffer for assay of alkaline phosphatase in serum. We evaluated N-methyl-D-glucamine (MEG) as a buffer for assay of alkaline phosphatase (ALP; EC 3.1.3.1) and compared the MEG-based assay with the current International Federation of Clinical Chemistry Reference Method for ALP (IFCC/RM/ALP), in which 2-amino-2-methyl-1-propanol (AMP) is the pH buffer. The ALP assay in MEG at 30 and 37 degrees C shows excellent correlation with the IFCC/RM/ALP at 30 degrees C, but yields proportionately higher ALP activities (8.2% at 30 degrees C and 57% at 37 degrees C). ALP is unstable in both MEG and AMP at 37 degrees C. Serum incubated in MEG undergoes a pH-dependent biphasic loss of ALP activity: an initial rapid 5% loss after 1 min of incubation and a 10% loss per hour thereafter. A similar pattern was seen for incubation with AMP. The use of a serum-initiated reaction (no preincubation of enzyme with buffer) eliminated the early loss in activity. The addition of the metal ion buffer N-hydroxyethylethylenediaminetriacetic acid, along with low concentrations of Zn and Mg, as used in the IFCC/RM/ALP, reduced the slow loss in activity over time, as did decreasing the reaction temperature to 30 degrees C, but had no effect on the early rapid decay in activity seen in the first minute. Moderate transphosphorylation (45%) and nonenzymatic hydrolysis (3.3 U/L) were observed with MEG under the conditions of the assay (37 degrees C). A comparison of different lots of MEG from two manufacturers showed no significant difference in ALP activities.


Decreases in albumin/creatinine and N-acetylglucosaminidase/creatinine ratios in urine samples stored at -20 degrees C. The effects of storage for 6 months or 2 years at -20 degrees C were studied in urine samples from Type II diabetic patients by assaying albumin by immunoturbidity, N-acetylglucosaminidase (EC 3.2.1.30) by methoxynitrovinylphenol release, and creatinine by the Jaffé method. There were significant decreases (P < 0.001) in albumin/creatinine ratios from 1.14 (0.63-2.98) to 0.83 (0.32-2.12) g/mol (median + interquartile ranges) after 6 months (n = 97), and from 1.64 (0.74-5.72) to 1.00 (0.37-4.54) g/mol after 2 years (n = 89). The percentage of samples with results below the detection limit of the albumin assay (2 mg/L) increased from 5% to 21% after 6 months and from 0% to 34% after 2 years. N-Acetylglucosaminidase/creatinine ratios decreased (P < 0.001) from 520 (358-832) to 380 (263-695) U/mol after 6 months and from 520 (330-865) to 258 (82-462) U/mol after 2 years. The effect of storage was greater in samples with concentrations in the normal range (< 2.5 g/mol for albumin/creatinine, < 500 U/mol for N-acetylglucosaminidase/creatinine). Samples with albumin concentrations more than twice the normal range were still detected as abnormal after storage at -20 degrees C; e.g., 18% were > 5 g/mol (albumin/creatinine) initially, with 17% > 5 g/mol after 6 months of storage. We therefore recommend storage of urine samples at 4 degrees C for no longer than 7 days before assay.


Measurement of cyclosporine by liquid chromatography and three immunoassays in blood from liver, cardiac, and renal transplant recipients. In an effort to replace HPLC for whole-blood determination of cyclosporine (CsA), we compared HPLC with radioimmunoassay (RIA; INCSTAR, Cyclo-Trac SP assay), fluorescence polarization immunoassay (FPIA; Abbott TDx), and in-house modified enzyme-multiplied immunoassay technique (EMIT; Syva Co.). For blood samples from 200 various transplant recipients, RIA = 1.262 (HPLC) - 8.16, r = 0.983; FPIA = 1.200 (HPLC) + 19.90, r = 0.981; and EMIT = 1.038 (HPLC) + 11.28, r = 0.985. For segregation by transplant type, RIA, FPIA, and EMIT demonstrated positive biases of 27%, 12%, and 3%, respectively, for liver transplant recipients (n = 50) when compared with HPLC. Heart transplant recipients (n = 50) gave positive bias values of 23%, 14%, and 4% for RIA, FPIA, and EMIT, respectively. Adult renal transplant recipients (n = 50) demonstrated positive bias values of 30%, 31%, and 0% for RIA, FPIA, and EMIT, respectively. For pediatric renal transplant recipients (n = 50), positive biases of 40%, 31%, and 9% were obtained for RIA, FPIA, and EMIT, respectively. We conclude that the modified EMIT represents the best replacement for HPLC.


Bone-resorption markers galactosyl hydroxylysine, pyridinium crosslinks, and hydroxyproline compared. We compared the clinical performances of four bone-resorption (BR) assays (hydroxyproline, HYP; galactosyl hydroxylysine, GHYL; deoxypyridinoline, DPD; and pyridinoline, PYD) in subjects with different BR rates: normal (adult men and premenopausal women), mildly increased (postmenopausal osteoporotic women), high (Paget disease patients), and very high (children). The discrimination power (Z score) and the accuracy (estimated by receiver-operating characteristic analysis) for GHYL, DPD, and PYD were compared with those for HYP. Discrimination power and accuracy were similar for high- and very-high-BR groups for all four assays. However in the mildly increased-BR group, DPD, GHYL, and PYD showed a higher discrimination power and accuracy than did HYP. The clinical performances of HYP, DPD, GHYL, and PYD are comparable for large changes in BR. For modest changes, DPD, GHYL, and PYD are more accurate and have a higher discrimination power than does HYP.


A new case of IgE myeloma.

A new case of IgE myeloma. We report a case of IgE myeloma in a 78-year-old woman who presented with bone pain in the shoulder and hip and progressive weakness. Except for hypercalcemia, routine chemistry values were within normal limits. Hemoglobin was decreased and the leukocyte count slightly increased. Plasma cells were not observed in the peripheral blood. Serum protein electrophoresis showed a monoclonal protein in the beta-globulin fraction. Immunofixation confirmed the presence of an IgE kappa monoclonal protein. A bone marrow biopsy revealed an interstitial and nodular infiltration of abnormal plasma cells comprising 60% of nucleated cells present. Skeletal roentgenograms and bone scans of this patient showed osteolytic lesions and osteopenia of the thoracic and lumbar spine and osteolytic destruction of the right half of the sacrum. Flow-cytometric analysis of mononuclear cells isolated from peripheral blood showed that 15% of the lymphocytes bound IgE. Using cell-surface markers, we identified 45% of the IgE-positive cells as natural killer cells. Similar results have been found in other diseases marked by increased IgE. The clinical, radiological, and laboratory findings for this patient are compared with previously reported cases of IgE and other types of myeloma.


A hyaluronan-loading test applied to patients with liver and joint diseases. The serum hyaluronan disappearance data, after an intravenous bolus injection of hyaluronan, were evaluated in terms of model-based parameters. The loading test was performed in 10 healthy persons (basal serum hyaluronan concentration, C0, 24.9 +/- 8.9 micrograms/l [mean +/- S.D.]), 6 patients with joint disease (62.3 +/- 41.1 micrograms/l) and 19 patients with liver disease (206 +/- 214 micrograms/l). The highest maximum Michaelis-Menten elimination rate (Vmax = 287 +/- 86 micrograms/min) was found in patients with joint disease, significantly higher than in healthy persons (Vmax = 179 +/- 16, P = 0.0015) and in patients with liver disease (Vmax = 149 +/- 59, P = 0.0002). C0 and Vmax were evaluated as discriminants for assessment of residual liver function. In patients with liver disease C0 correlated with liver function score (r = 0.875, P < 0.0001) and serum albumin concentration (r = -0.813, P < 0.0001). The Vmax parameter did not correlate with conventional liver function tests or with the liver score but a significantly negative correlation of Vmax with C0 was found in patients with liver disease. A combination of the C0 level and the Vmax parameter was found to discriminate between healthy persons, patients with joint disease and patients with liver disease and should be of benefit in separating patients, with or without elevated serum hyaluronan levels, into groups having increased influx or reduced elimination, respectively, of circulating hyaluronan.


Cellulose acetate electrophoretic determination of bone alkaline phosphatase activity in healthy subjects and diabetic patients with and without osteopenia. We adapted the electrophoretic method of bone alkaline phosphatase (ALP) determination using neuraminidase from Vibrio cholerae to separate bone and liver ALP on cellulose acetate membrane. Treatment of separator plus serum (1:8, neuraminidase 111 U/l in final) for 10 min at room temperature (25 +/- 1 degree C) and subsequent electrophoresis made it possible to quantify bone ALP activity simply and rapidly. The precision of the data was at the level of CV of 1.6% (within-day) and 4.7% (day-to-day), with recovery rates of 97-103%. The normal range of bone ALP activity depended on age and sex. Seventy-eight diabetes mellitus (DM) patients, excluding those with renal failure, were divided into two groups of those with and without osteopenia with matching of age (+/- 3 years) and sex. Bone ALP (P < 0.001) and total ALP (P < 0.05) activities and urine calcium/creatinine ratio (P < 0.05) were significantly higher in DM with osteopenia than in DM without osteopenia. Therefore, bone formation and absorption may be accelerated in DM with osteopenia in comparison with DM without osteopenia.


CA 19-9 in non-neoplastic liver diseases. A clinical and laboratory study. CA 19-9 is a tumor marker with frequent false-positive results in pancreatic and hepatobiliary diseases. This study was carried out to evaluate the behaviour of CA 19-9 in 159 patients with benign diffuse hepatic disease, 85 cirrhotics and 74 non-cirrhotics, who underwent a thorough clinical and laboratory evaluation. CA 19-9 was correlated with numerous clinical and biochemical features of liver diseases: bilirubin and alkaline phosphatase activity were the most reliable predictors of the CA 19-9 concentrations. There were abnormal concentrations of CA 19-9 in 34.6% of the 159 patients and in 47.1% of the 85 cirrhotics. Because of the large number of abnormal values and the high concentrations attained in some of them, the cut-off used in patients with diffuse hepatic disease needs to be set at more than twice the basal level, thus allows only 10% of false positives. Even higher values are required for cirrhotic or icteric patients. The results indicate that cholestasis plays an important role in causing the raised CA 19-9 in these patients, although there were also abnormal concentrations in normobilirubinemic patients.


Arginase, a new marker of mammary carcinoma. Activities of arginase, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase were determined in sera obtained in a group of healthy women, women with verified carcinoma of the breast, benign mastopathy, a group of patients with carcinoma of various organs and a group of patients with acute viral hepatitis. Preoperative values of serum arginase activity in patients with breast carcinoma were up to 4-fold those found in healthy women. Sensitivity of the test was 86%. After the surgery, the activity decreased abruptly during the first week and normalised within 15-30 days. In benign diseases of the breast, the activity of arginase was normal. Serum arginase activity is raised in both benign and malignant liver diseases, however, the quotients alanine aminotransferase/arginase, aspartate aminotransferase/arginase and alkaline phosphatase/arginase differ significantly. Thus, use of alanine aminotransferase/arginase quotient implies a high degree of confidence in differentiating between increased arginase activity in mammary carcinoma (alanine aminotransferase/arginase = 0.572 +/- 0.278) and high arginase activity in hepatitis (alanine aminotransferase/arginase = 12.226 +/- 1.822).


Identification of urinary metabolites of (+/-)-2-(p-isobutylphenyl)propionic acid (Ibuprofen) by routine organic acid screening. Ibuprofen [(+/-)-2-(p-isobutylphenyl)propionic acid] has recently been introduced as a pediatric anti-inflammatory agent. To determine how this agent interferes with urine organic acid analysis, an important pediatric investigation, we have analyzed urine from two subjects pre- and post-Ibuprofen dosage and two subjects on chronic Ibuprofen therapy. A distinctive pattern of drug interference on the organic acid profile was detected. There were three major components, corresponding to unmetabolized Ibuprofen and to the oxidation products hydroxy Ibuprofen and carboxy Ibuprofen. Therefore, the major mechanism of Ibuprofen metabolism appears to be microsomal, although mitochondrial and peroxisomal routes cannot be excluded. Laboratories carrying out routine organic acid analysis should be aware of the nature and magnitude of the organic aciduria caused by Ibuprofen.


Intestinal type alkaline phosphatase hyperphosphatasemia associated with liver cirrhosis. Hyperphosphatasemia due to increased intestinal type serum alkaline phosphatase was noted in a 48-year-old male who had asymptomatic liver cirrhosis. The alkaline phosphatase activity in the serum was 828 U/l (our reference range in adults: 57-194 U/l), 94% of which was of the intestinal type as measured by an immunoprecipitation method. The intestinal component of alkaline phosphatase was separated into two major and some minor components using electrophoresis and isoelectrofocusing. One of the major components had similar mobility to that of a standard intestinal enzyme purified from adult intestine. The components were heat-labile and neuraminidase-resistant. Serial lectin affinity chromatography, however, indicated that sugar chain compositions of the alkaline phosphatase were different from those of the standard tissue intestinal enzyme. These results and further enzymological studies suggest that the patient's serum alkaline phosphatase basically consisted of several intestine-like isoforms.


Purification of electron transfer flavoprotein from pig liver mitochondria and its application to the diagnosis of deficiencies of acyl-CoA dehydrogenases in human fibroblasts. Medium chain acyl-CoA dehydrogenase (MCAD) and long chain acyl-CoA dehydrogenase (LCAD) deficiency are defects of mitochondrial beta-oxidation. The method of choice to measure specifically acyl-CoA dehydrogenase activity in human tissues uses purified electron transfer flavoprotein (ETF). We describe a simple and optimized method of purification allowing isolation of ETF with a degree of purity never reported so far. An assay for acyl-CoA dehydrogenase activity in cultured skin fibroblasts was developed using microquantities of electron transfer flavoprotein and substrate. MCAD deficiency was demonstrated in fibroblasts from nine patients and LCAD deficiency in fibroblasts from two patients.


The evaluation of patients with a suspected pituitary microadenoma: computer tomography compared to magnetic resonance imaging. To perform a prospective study to compare the ability of magnetic resonance imaging (MRI) and computer assisted tomography (CT) to predict the position of a tumour within the pituitary fossa and to assess the clarity of the image generated by the two modalities in patients with suspected pituitary microadenomas. Nineteen patients were investigated with CT and MRI scans and underwent trans-sphenoidal exploration of the pituitary fossa. Both scans were assessed blind and independently by three neuroradiologists, A, B, and C; A and B examined the CT scans and B and C the MRI scans. The predicted site was then compared to the size and position of the tumour at surgery. Twenty-five patients were recruited and had both CT and MRI scans, 19 subsequently underwent trans-sphenoidal exploration of the pituitary fossa and formed the study population. The joint opinions of A and B of the CT scans were correct in 10/19, and those of B and C of the MRI scans were correct in 17/19 cases, P = 0.008. Observer agreement was used to assess the clarity of the image. A and B agreed about the site of the tumour on the CT scan in 14 of 19 cases (Kappa statistic 0.556); B and C agreed on the MRI scans in 19/19 cases (Kappa statistic 1), P = 0.025. After the exclusion of five cases found to be macroadenomas at surgery, the joint opinions of A and B of the CT scans were correct in 8/14, and those of B and C of the MRI scans were correct in 12/14 cases, P = 0.133. A and B agreed about the site of the tumour on the CT scan in 12/14 cases (Kappa statistic 0.653), B and C agreed on the MRI scans in 14/14 cases (Kappa statistic 1, no significant difference). In the assessment of suspected pituitary microadenomas MRI is the imaging technique of choice in view both of its greater diagnostic accuracy and the avoidance of radiation exposure.


Age related changes in follicle stimulating hormone, luteinizing hormone, oestradiol and immunoreactive inhibin in women of reproductive age. In women over the age of 45 years with continuing regular menstrual cycles, follicular phase FSH levels rise without an accompanying change in LH. We determined the effect of increasing age in women with regular cycles on the serum levels of FSH, LH, immunoreactive inhibin, progesterone and oestradiol. Single blood samples were taken during the early follicular phase (days 4-7) and again in the midluteal phase (3-12 days before the next menses) of the menstrual cycle. Regularly cycling women aged 21-49 years participated in the study (and were grouped into four groups: 20-29, 30-39, 40-44 and 45-49 years in the follicular phase and three groups: 20-29, 30-39 and 40-49 years in the luteal phase. Serum levels of FSH, LH, oestradiol, progesterone and immunoreactive inhibin were measured from the blood samples obtained. Follicular phase Mean follicular phase levels of immunoreactive inhibin were significantly lower in the 45-49 year age group (P less than 0.05) than in the younger age groups (128 U/l in the 45-49 year age group vs 239, 235 and 207 U/l in the 20-29, 30-39, 40-44 year age groups respectively), while mean FSH levels were significantly higher in the 45-49 year age group (P less than 0.05, 13.0 IU/l in the 45-49, 4.9, 5.5 and 5.2 IU/l in the 20-29, 30-39 and 40-44 year age groups respectively). Mean oestradiol levels in the 45-49 year age group were significantly lower only when compared to age group 30-39 years (P less than 0.05, 130 vs 210 pmol/l). There was no significant difference in oestradiol levels between the 45-49 year age group and the 20-29 and 40-44 year age groups. LH levels did not differ significantly across age groups. There was also a significant negative correlation between serum immunoreactive inhibin and FSH (r = -0.45, P less than 0.05) and between oestradiol and FSH (r = -0.35, P less than 0.05). There was a significant negative relationship between immunoreactive inhibin and age (r = -0.46, P less than 0.05). For every 10-year increase in age, average immunoreactive inhibin decreased by an estimated 49.3 U/l. As age increased, average FSH levels exhibited a two-phase linear increase with the change-point estimated at 42.97 (1.42) (estimate (SE)) years. Prior to 42.97 years, FSH barely changed; after 42.97 years there was a significant (P less than 0.05) increase in FSH as age increased. Oestradiol levels did not change significantly until an estimated 37.9 years of age, but then decreased significantly (P less than 0.05) with increasing age. Luteal phase Levels of FSH, LH, serum immunoreactive inhibin, oestradiol and progesterone fell slowly with increasing age. There was a significant correlation between serum immunoreactive inhibin with progesterone (r = 0.41, P less than 0.05) but there was no correlation between serum immunoreactive inhibin LH or FSH. The results are consistent with a role for serum immunoreactive inhibin, in addition to oestradiol, in the regulation of FSH during the follicular phase of the menstrual cycle as a function of increasing age. This is postulated to reflect diminished folliculogenesis as age progresses with the known decline in the numbers of primordial follicles in the ovary as the menopause approaches.


Growth hormone binding protein in Werner's syndrome. GH and growth hormone binding protein in Werner's syndrome were investigated to elucidate their relation to the short stature. The levels of GH binding protein and GH response to insulin-induced hypoglycaemia were determined. GH binding protein levels and its Scatchard analysis in Werner's syndrome were compared with those in normal subjects. Three patients with Werner's syndrome (one man aged 45 years and two women aged 39 and 38 years) and 41 normal subjects (18 men and 23 women aged 39.3 +/- 5.5 years, mean +/- SD) were studied. GH binding protein levels were determined using an Ultrogel AcA44 minicolumn and GH levels were measured by a highly sensitive enzyme immunoassay. Two out of three patients with Werner's syndrome had GH binding protein levels above the mean +/- 2SD value in normal subjects. GH secretion was impaired in Werner's syndrome as judged by the low GH response to insulin-induced hypoglycaemia. Elevated GH binding protein levels may lead to an increase in the bound form of GH, which is probably less bioactive, resulting in growth failure in association with the impaired GH secretion in Werner's syndrome.


Preserved thyroidal secretion of thyroxine in acromegalic patients with suppressed hypophyseal secretion of thyrotrophin. We have assessed the mechanisms which maintain euthyroidism in acromegalic patients despite the suppression of thyrotrophin (TSH) secretion. Fourteen untreated patients with acromegaly were analysed. Ten patients were also studied after pituitary surgery. Thyroid hormones, growth hormone (GH), insulin-like growth factor-I (IGF-I) and thyroidal uptake of radioactive iodine, thyrotrophin releasing hormone (TRH) test and basal metabolic rate (BMR) were measured before and after pituitary surgery. Nine patients had palpable goitres. The TSH response to TRH stimulation was suppressed in eight patients, who maintained normal serum levels of total T3, T4 and free T4. The patients with normal TSH response had lower levels of free and total T4 than controls. The response of TSH to TRH correlated inversely with the serum level of total and free T4, and also with the plasma level of IGF-I (r = -0.74, P less than 0.05, n = 9). After pituitary surgery, the serum levels of total and free T4 were elevated for at least up to 6 months, with a decrease in the T3/T4 ratio and the BMR. GH may have a direct stimulatory action on the thyroid secretion of T4 possibly via increased IGF-I, despite suppressed TSH secretion. The post-operative elevation of serum T4 suggests the persistent secretion of T4 from the thyroid gland, in spite of instantaneous normalization of the accelerated conversion of T4 to T3, even after reduction of excess GH secretion.


Failure of somatostatin analogue to control Cushing's syndrome in two cases of ACTH-producing carcinoid tumours. To determine the effectiveness of somatostatin analogue (octreotide) in controlling hypercortisolism in two patients with ectopic ACTH-producing carcinoid tumours, and to review the literature. The two patients were treated with octreotide administered by subcutaneous injection, for 5 days with 150 micrograms three times daily and for 7 days with 100 micrograms three times daily respectively. They were subsequently treated with oral metyrapone 250 mg three times daily. Patient 1 had a metastatic carcinoid tumour but the primary was not identified. Patient 2 had a pulmonary carcinoid. Cushing's syndrome due to ectopic ACTH syndrome was established by demonstration of failure of cortisol suppression by dexamethasone, elevated ACTH levels, and immunoperoxidase staining for ACTH within the tumours. Urinary free cortisol (UFC) was measured on consecutive days during treatment with octreotide. Serum cortisol and ACTH levels were taken daily in patient 2, and on days 0 and 3 in patient 1. Patient 1 had a baseline 24-hour urinary free cortisol of 5340 nmol/24 h, serum cortisol of 915 nmol/l, and serum ACTH of 163 ng/l (ACTH ng/l x 0.23 = pmol/l). After 3 days of octreotide, serum cortisol was 782 nmol/l and ACTH 164 ng/l. Twenty-four hour urinary free cortisol was 4136 nmol/24 h after 7 days of treatment. Metyrapone, however, resulted in a rapid fall in urinary free cortisol to 290 nmol/24 h, with marked clinical improvement. Patient 2 had a baseline 24-hour urinary free cortisol of 2520 nmol/24 h, serum cortisol of 747 nmol/l, and ACTH of 103 ng/l. Urinary free cortisol rose to 2970 nmol/24 h on day 6 of treatment with octreotide. Serum cortisol and ACTH levels fell slightly to 611 nmol/l and 70 ng/l respectively. On changing to metyrapone, the urinary free cortisol fell to 821 nmol/24 h in 4 days. Octreotide failed to significantly reduce 24-hour urinary free cortisol, serum cortisol and ACTH in the two patients reported. We conclude that it should probably not be regarded as primary treatment for control of hypercotisolism in patients with ACTH-producing carcinoids, but reserved as adjunctive therapy.


Efficacy of a new long-acting injectable form of bromocriptine in hyperprolactinaemic patients. The objective of this study was to assess the relationship of different doses of a long-acting bromocriptine preparation (Parlodel LAR) to the degree and duration of PRL suppression. We also measured circulating bromocriptine levels and altered tolerability of the drug. A double-blind randomized study of three different doses 25, 50 and 100 mg of Parlodel LAR. Twenty-one female patients (seven patients/dose) with both tumoral and non-tumoral hyperprolactinaemia. After a single injection of Parlodel LAR 25, 50 or 100 mg, serum PRL and plasma bromocriptine levels were assessed during a follow-up of 60 days together with changes in clinical symptoms and signs of hyperprolactinaemia. Serum PRL levels normalized in 19 of 21 patients. The suppression of PRL secretion lasted 28 days in four of seven patients treated with either 25 or 50 mg Parlodel LAR and in five of seven patients who received Parlodel LAR 100 mg. In five of seven patients treated with the 100 mg dose, serum PRL levels were still within the normal range on day 60. Plasma bromocriptine levels remained therapeutically active for 28 days in all three groups. On day 60 they were within the therapeutic range only in the 100 mg group. Clinical data show a rapid disappearance of symptoms and signs of hyperprolactinaemia. Adverse events were mostly mild and transient. These data support the excellent efficacy and good tolerability of Parlodel LAR in patients with hyperprolactinaemia.


Age-related changes in bone density, serum parathyroid hormone, calcium absorption and other indices of bone metabolism in Chinese women. To study the age-related changes in bone density, serum parathyroid hormone, calcium absorption and other indices of calcium metabolism in Chinese women who habitually have a low calcium intake. Cross-sectional study. One hundred and fifty-six healthy Chinese women aged 20-83 years. None were on any medication or vitamin supplements. Subjects over the age of 60 years were all living in a hostel; younger subjects were nurses or subjects attending a family clinic for minor illnesses. Fasting blood and urine samples were collected for biochemical measurements and calcium absorption was measured using 45Ca by the method of Marshall and Nordin. Bone density was measured by dual energy X-ray densitometry (Norland X R20 X-ray bone densitometer) at the left hip and lumbar spine. Serum parathyroid hormone was measured by a chemiluminometric assay. Plasma ionized calcium concentration, alkaline phosphatase, bicarbonate, plasma creatinine and serum B2 microglobulin were significantly higher in the elderly than in the young, whereas plasma phosphate and the anion gap were higher in the young. Urinary excretion of calcium, phosphate and hydroxyproline were all higher in older women. Plasma parathyroid hormone concentration was positively correlated with age even after taking into account the decline in renal function (as indicated by the rise in B2 microglobulin (r = 0.506, P less than 0.001). Serum concentrations of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were not lower in the older women. Fractional calcium absorption from an oral load of radiolabelled calcium was significantly lower in the older women and 37% of the older women were below the 2.5 percentile found in the younger women. Bone density measured by dual energy X-ray densitometry was also significantly lower in the elderly. In Chinese women there is an increase in PTH and a decrease in calcium absorption with age in spite of the presence of normal vitamin D metabolites.


Circulating immunoreactive inhibin and testosterone levels in men with critical illness. We aimed to concurrently characterize serial changes in circulating immunoreactive inhibin (irINH) and testosterone (T) as reflections of Sertoli and Leydig cell responses to acute critical illness in man. Blood samples were drawn within 24 hours of admission to an Intensive Care Unit and at weekly intervals thereafter for up to 4 weeks while the patient remained in Intensive Care Unit or after discharge to a general ward. We studied 13 male subjects with critical illness requiring intensive therapy. Plasma levels of irINH, T, LH, FSH and sex hormone binding globulin (SHBG) were analysed in relation to (i) the severity of illness as indicated by a sepsis score, acute physiology and chronic health evaluation score, and reverse triiodothyronine (rT3) levels and (ii) the outcome of illness as determined by discharge from Intensive Care Unit and the two-month mortality. Overall irINH levels remained normal and correlated negatively with rT3 (r = -0.63, P = 0.001) but not with sepsis, acute physiology and chronic health evaluation score, or gonadotrophin levels. Neither admission nor serial irINH levels significantly distinguished between the different clinical outcomes. In contrast, T levels were depressed and inversely correlated with both sepsis and acute physiology and chronic health evaluation scores (P less than 0.02), and positively with gonadotrophins (P less than 0.01), but not rT3 levels. Men eventually discharged from the Intensive Care Unit showed a rise, while those remaining showed a fall, in T levels (P = 0.02, time-course interaction). Similarly, T levels were lower in patients who died than in survivors, despite the comparable T levels on admission (P = 0.02, time-course interaction). Despite the fall in T levels, gonadotrophin levels remained inappropriately in the eugonadal range but higher in men who were discharged from Intensive Care Unit (P = 0.02, time-course interaction). FSH but not LH levels were correlated with sepsis score (P = 0.02) but not acute physiology and chronic health evaluation score or rT3. Sertoli cell function as judged by circulating irINH levels is much less affected by acute critical illness than is Leydig cell function as judged by circulating T levels. The suppressive effect of acute critical illness on Leydig cell function is consistent with a hypothalamic-pituitary lesion.


Social, educational and vocational status of 48 young adult females with gonadal dysgenesis. The purpose of this study was to evaluate the educational, vocational and social function of young adults with gonadal dysgenesis. Forty-eight female patients with gonadal dysgenesis (17, 45XO; 26, 45XO/46XX or other mosaics; and five pure gonadal dysgenesis) followed by our multidisciplinary team from childhood, were re-evaluated in adult age. Mean age +/- SD at diagnosis was 11.4 +/- 5.0 years and mean age at the time of survey was 29.6 +/- 6.3 years. The mean final height for the patients was 145.5 +/- 8.3 cm (range 134-170 cm). Mean verbal IQ (WISC-R) for the 39 subjects tested was 101.4 +/- 20.7 and mean performance IQ was 86.8 +/- 17.7. No difference in verbal IQ levels was found between the various karyotype groups. Twenty-five had an academic education. All were employed except for one housewife; 37 work in white collar professions. A significant correlation was found between verbal IQ and education (P = 0.005) and between verbal IQ and profession (P = 0.005). Twenty-three served in the army. Fourteen are married: three have an adopted child and two a child born after in vitro fertilization (IVF); others are waiting for IVF or adoption. Five patients had some form of psychiatric problem that required psychiatric or psychological treatment in the past (two had transitory anorexia nervosa and three behavioural problems). Sixty-three per cent reported having wide and satisfactory social relations but limited to female friends. Positive adjustment in the professional area and fair adjustment in the social area were not related to physical stigmata or to any other independent variable tested, but rather to intellectual ability and a high degree of achievement motivation.


Changes in growth hormone concentrations during puberty in adolescents with insulin dependent diabetes. To document the changes in pulsatile growth hormone secretion in diabetic adolescents during puberty, and to investigate their relationship to both metabolic control and stature. Auxological parameters, overnight growth hormone secretion, fasting IGF-I, hourly glucose and metabolic control were assessed in a group of adolescents with diabetes. Fifty-two diabetic adolescents (28 males and 24 females) at different pubertal stages and with varying degrees of metabolic control were studied. Ten of those with poor diabetic control were studied on two occasions. Height and weight measurements, pubertal staging, growth velocity data and bone age estimation were obtained on all the patients. Overnight growth hormone profiles (Pulsar program analysis), glycosylated haemoglobin and fasting IGF-I were performed on all the subjects. Hourly overnight glucose measurements were also obtained on the ten subjects who had two overnight growth hormone studies. For the whole diabetic growth, GH area under curve (AUC) was maximal in late puberty (pubertal stage 4), and was paralleled by maximal GH peak amplitude. No relationship between GH-AUC and metabolic control was demonstrated. No difference in GH parameters was demonstrated between the male and female subgroups. The relationship between growth hormone secretory parameters and stature was not significant. However, GH-AUC was significantly correlated with growth velocity in the males but not the females. The pattern of GH secretion in adolescents with diabetes parallels that seen in normal adolescents during puberty, with increases in GH concentration associated with increased GH pulse amplitude. The degree of metabolic control had no effect on this pattern and there was no relationship between GH secretory parameters and stature.


Abnormalities of intermediary metabolism following a gestational diabetic pregnancy. The aim of this study was to compare intermediary metabolism in glucose tolerant women with previous gestational diabetes and control women without a history of gestational diabetes. Fifteen women with previous gestational diabetes and 15 controls individually matched for race, age and body mass index were included. Only subjects with normal glucose tolerance were included in this study. Plasma glycerol, 3-OH butyrate, non-esterified fatty acids (NEFA), glucose and insulin were measured fasting and following a 75 g oral glucose load. The women with previous gestational diabetes and their matched controls were of similar racial origin, age and body mass index. There was no difference between those with previous gestational diabetes and controls in fasting glycerol, 3-OH butyrate, NEFA, glucose or insulin concentrations. Following the oral glucose load, glycerol, 3-OH butyrate and NEFA concentrations fell in both groups. However, compared with controls at 120 minutes, those with previous gestational diabetes had significantly higher concentrations of glycerol (median 57, range 24-216 vs 27, range 8-89 mumols/l, P less than 0.005) and 3-OH butyrate (9, range 1-18 vs 5, range 2-11 mumols/l, P less than 0.01). NEFA concentrations were similar in the two groups. Despite similar glucose concentrations during the oral glucose tolerance test the insulin response during the first 60 minutes following oral glucose was significantly reduced in the subjects with previous gestational diabetes when compared with controls (insulin area, 0-60 minutes; 2172, range 788-4767 vs 2830, range 996-4784 pmol min/l, P less than 0.05). Women with a previous history of gestational diabetes, despite having normal glucose tolerance, have defective insulin release with resultant abnormalities of intermediary metabolism.


The importance of adequate serum ferritin levels during oral cyproterone acetate and ethinyl oestradiol treatment of diffuse androgen-dependent alopecia in women. To evaluate the treatment with cyproterone acetate and ethinyl oestradiol of diffuse androgen-dependent alopecia in women. Twenty women were treated for a period of 12 months in a reverse sequential regimen employing cyproterone acetate (CPA, 50 mg once daily from Day 5 to Day 15) and ethinyl oestradiol (EE2, 30 micrograms once daily from Day 5 to Day 24 of the menstrual cycle), and 20 were left untreated and acted as controls. Half of each group had serum ferritin concentrations above or below 40 micrograms/l. Forty Caucasian premenopausal women aged between 18 and 47 years, presenting with diffuse androgen-dependent alopecia, were enrolled into this study. Hair variables were assessed initially by the unit area trichogram and again in the same sites 12 months later. Biochemical investigations were performed before treatment and after 3, 6, and 12 months. In the treated group, a significant (P less than 0.01) mean increase in total hair density (hair/cm2) and meaningful hair density (non-vellus hair/cm2) was found in patients in whom the serum ferritin was above, but not below 40 micrograms/l. However, in the control group a significant (P less than 0.05) mean decrease in total hair density and meaningful hair density was observed after 12 months. No correlation between serum ferritin levels and the degree of hair loss within this group could be established. Patients treated with the anti-androgen cyproterone acetate and ethinyl oestradiol respond best when serum ferritin is above 40 micrograms/l.


Nelson's syndrome following partial pituitary microadenomectomy and pregnancy. We report for the first time the development of Nelson's syndrome in a patient who had previously undergone unsuccessful pituitary microadenomectomy and subsequently bilateral adrenalectomy. The removal of a 3-mm portion of a microadenoma did not protect against the development of Nelson's syndrome within 3 years which was closely related to pregnancy, a previously noted association. This report suggests that the development of Nelson's syndrome depends not on the size of the initial pituitary tumour but rather on its individual potential for proliferating once free of the inhibitory effects of glucocorticoid excess.


Postprandial thermogenesis is reduced in polycystic ovary syndrome and is associated with increased insulin resistance. In order to investigate the possible causes and effects of obesity in polycystic ovary syndrome resting energy expenditure, postprandial thermogenesis and insulin resistance were measured in 14 polycystic ovary syndrome subjects and in 14 controls. A cross-sectional study of a selected group of patients was performed. Seven of the PCOS subjects were obese and seven lean. Controls were individually matched for age, race, weight, body mass index (BMI) lean body mass and percentage fat. The obese, but not lean, polycystic ovary syndrome subjects had a greater waist:hip ratio than controls (median (range) obese PCOS 0.865 (0.823-0.960) vs obese control 0.804 (0.823-0.940), P less than 0.025). Metabolic rate was measured by continuous indirect calorimetry and insulin sensitivity was assessed by a short insulin tolerance test. The resting energy expenditure (REE) was similar in PCOS subjects and controls (median (range), 6796 (5489-7774) vs 6833 (4893-8492) kJ/day). REE correlated with LBM in the PCOS group (r = 0.83, P less than 0.00) and the control group (r = 0.82, P less than 0.001). Postprandial thermogenesis was reduced in polycystic ovary syndrome (obese: median 45.4 (range 33.6-100.0) vs 86.5 (67.2-109.2) kJ (P less than 0.05); lean: 79.4 (73.5-108.4) vs 89.9 (76.0-109.2) kJ (P less than 0.05). Fasting insulin (9.7 +/- 3.6 vs 4.4 +/- 0.8 mU/l, P less than 0.05) and postprandial incremental insulin rise (163 +/- 31 vs 116 +/- 15 mU/l, P less than 0.025) were higher in polycystic ovary syndrome. Insulin sensitivity was reduced in polycystic ovary syndrome (obese: median 136 (range 92-169) vs 173 (109-225) mumol/l/min (P less than 0.05); lean: 161 (138-225) vs 194 (161-253) mumol/l/min (P less than 0.05)). The reduction in insulin sensitivity correlated with the reduced postprandial thermogenesis in the polycystic ovary syndrome group (r = 0.75, P less than 0.01). These results confirm previous reports of hyperinsulinaemia and insulin resistance in polycystic ovary syndrome. Furthermore, polycystic ovary syndrome subjects have a reduced postprandial thermogenesis which is related statistically to the reduced insulin sensitivity. The decreased postprandial thermogenesis may predispose women with polycystic ovary syndrome to weight gain.


Increased free testosterone but normal 5 alpha-reduced testosterone metabolites in obese premenopausal women. The aim of the study was to investigate whether the absence of increased 5 alpha-reductase activity explained the absence of hirsutism in premenopausal obese women with increased free testosterone (FT) levels. As in hyperandrogenicity there generally exists evidence for increased 5 alpha-reductase activity, we measured, as parameters of 5 alpha-reductase activity, plasma levels of 5 alpha-androstane-3 alpha,17 beta-diol glucuronide (ADG) and androsterone glucuronide (ADTG) as well as their precursor levels in obese women without hirsutism, obese hirsute women, non-obese hirsute women, and non-obese, non-hirsute women. Eighty-two premenopausal women (20-45 years old) were studied, in four age matched groups: 39 controls, 18 obese without hirsutism, 11 non-obese hirsute and 14 obese hirsute women. Blood samples were taken between days 5 and 7 of the menstrual cycle. Steroid hormone levels were measured by radioimmunoassay. Free testosterone levels were measured by equilibrium dialysis. Compared to controls, mean free testosterone levels were increased (P less than 0.01) in obese, obese hirsute and hirsute patients, whereas mean DHEAS levels were increased in hirsute and obese hirsute (P less than 0.01), but not in obese, women. Mean androstanediol glucuronide levels were markedly increased in hirsute and obese hirsute patients (P less than 0.01), but not in obese women. Plasma androsterone glucuronide levels were increased in hirsute (P less than 0.01), in the normal range in obese hirsute, and decreased in obese women (P less than 0.01). These results show that, despite the presence of higher free testosterone levels, neither 5 alpha-reductase activity (as suggested by normal androstanediol glucuronide levels) nor adrenal androgen precursor levels (DHEAS) are increased in obese women without hirsutism.


17 beta-Oestradiol, androstenedione and inhibin levels in fluid from individual follicles of normal and polycystic ovaries, and in ovaries from androgen treated female to male transsexuals. The aim was to monitor carefully follicular growth arrest in polycystic ovaries by assay of hormones in individual follicles. Fluid from follicles less than or equal to 10 mm was obtained from ovaries of 16 regularly cycling women between days 1 and 12 of the follicular phase (controls, n = 120 follicles), polycystic ovaries of five women with polycystic ovary syndrome (n = 43), and polycystic ovaries from 14 long-term testosterone treated female to male transsexuals (n = 120). Fluid was assayed for oestradiol, androstenedione, and immunoactive inhibin. Luteinizing hormone, follicle-stimulating hormone, and testosterone levels were estimated in serum. Median serum LH was lower in transsexuals than in controls (P less than 0.05), and in polycystic ovary syndrome (P less than 0.01). Median serum testosterone was not significantly different between polycystic ovary syndrome and transsexuals, and was elevated in both groups as compared to controls (P less than 0.01). Oestradiol was present in all follicles obtained from polycystic ovaries of polycystic and transsexual patients, in which no follicle greater than 10 mm could be detected. In the three groups, between-patient differences in mean oestradiol, androstenedione, inhibin, and androstenedione/oestradiol ratio were significantly larger than expected in view of the variation between follicles within individuals. Taking into account this between-patient difference, no significant differences could be established between the three groups for all endocrine parameters. The percentage of presumed healthy follicles (androstenedione/oestradiol ratio less than or equal to 4) was 12% in controls, 17% in polycystics, and 14% in transsexuals, and was not significantly different between groups. The results may indicate that (1) abnormally high circulating androgen concentrations with or without elevated LH levels disturb the process of selection, and could therefore play a role in the pathogenesis of polycystic ovaries; (2) in polycystic ovaries from polycystic ovary syndrome and transsexual patients, aromatase activity is present in vivo in small antral follicles, and the proportion of presumed healthy follicles is not different from that encountered in normal ovaries; (3) oestradiol levels are not different between non-dominant follicles of normal and polycystic ovaries, suggesting that only enhancement of aromatase activity by FSH may be disrupted in polycystic ovaries, (4) because androstenedione levels are not different comparing follicles of normal and polycystic ovaries, hyperandrogenaemia in the syndrome seems to originate from the abnormally high number of cystic atretic follicles generally observed in polycystic ovaries; (5) marked variation in the endocrine follicular microenvironment within and between-women precludes pooling fluid from several follicles.


The effect of iodide on serum thyroid hormone levels in normal persons, in hyperthyroid patients, and in hypothyroid patients on thyroxine replacement. To clarify the duration and the extent of the antithyroid effect of iodides in hyperthyroidism, and to investigate whether iodides have an additional peripheral effect on the metabolism of thyroid hormones, as has been reported for some organic iodine compounds. The effect on the peripheral thyroid hormone levels of 150 mg of potassium iodide daily (equivalent to 114 mg of iodide) for 3-7 weeks was compared in 21 hyperthyroid patients and 12 healthy controls. A possible effect of iodide on the peripheral metabolism of thyroid hormones was investigated by assessing the serum levels of thyroid hormone in 12 hypothyroid patients on thyroxine replacement for 2 weeks. There were 21 thyrotoxic patients, 12 healthy hospital controls, and 12 patients with complete or near-complete hypothyroidism, on thyroxine replacement. The following were measured before and at weekly intervals after iodide administration: (1) pulse rate, (2) serum T4, (3) serum T3, (4) serum TSH, (5) serum thyroxine-binding capacity (TBC), (6) serum rT3, (7) serum thyroxine-binding globulin (TBG), (8) the free-T4 Index, calculated as T4/TBC. In the hyperthyroid patients serum T4, T3 and rT3 decreased, whereas serum thyroxine-binding globulin and thyroxine binding capacity increased. Serum T3, however, did not become completely normal in all cases. After 21 days, serum T4 and T3 started increasing again in some cases, but other patients remained euthyroid even after 6 weeks. In the normal controls there was a small but significant and consistent decrease in serum T4, T3 and rT3 and an increase in serum TSH. Finally, in the T4-treated hypothyroid patients there was no consistent change, except for an increase of serum T4 at 1 and 14 days and a decrease of serum TSH the first day. Iodides in hyperthyroidism have a variable and unpredictable intensity and duration of antithyroid effect. Their antithyroid effect is smaller in normal controls. They have no important effect on the peripheral metabolism of thyroid hormones.