A mouse model of familial porphyria cutanea tarda.

Approximately one-third of patients with porphyria cutanea tarda (PCT), the most common porphyria in humans, inherit a single mutant allele of the uroporphyrinogen decarboxylase (URO-D) gene. PCT associated with URO-D mutations is designated familial PCT. The phenotype is characterized by a photosensitive dermatosis with hepatic accumulation and urinary excretion of uroporphyrin and hepta-carboxylic porphyrins. Most heterozygotes for URO-D mutations do not express a porphyric phenotype unless hepatic siderosis is present. Hemochromatosis gene (HFE) mutations are frequently found when the phenotype is expressed. We used homologous recombination to disrupt one allele of murine URO-D. URO-D(+/-) mice had half-wild type (wt) URO-D protein and enzymatic activity in all tissues but did not accumulate hepatic porphyrins, indicating that half-normal URO-D activity is not rate limiting. When URO-D(+/-) mice were injected with iron-dextran and given drinking water containing delta-aminolevulinic acid for 21 days, hepatic porphyrins accumulated, and hepatic URO-D activity was reduced to 20% of wt. We bred mice homozygous for an HFE gene disruption (HFE(-/-)) to URO-D(+/-) mice, generating mice with the URO-D(+/-)/HFE(-/-) genotype. These animals developed a porphyric phenotype by 14 weeks of age without ALA supplementation, and URO-D activity was reduced to 14% of wt. These data indicate that iron overload alone is sufficient to reduce URO-D activity to rate-limiting levels in URO-D(+/-) mice. The URO-D(+/-) mouse serves as an excellent model of familial PCT and affords the opportunity to define the mechanism by which iron influences URO-D activity.

Accelerated development of uroporphyria in mice heterozygous for a deletion at the uroporphyrinogen decarboxylase locus.

Three weeks after a single dose of iron-dextran and Aroclor 1254, mice maintained continuously on delta-aminolevulinic acid supplemented drinking water showed significantly elevated levels of hepatic uroporphyrin and depressed (25% of normal) uroporphyrinogen decarboxylase (URO-D) activity. Depressed URO-D activity was paralleled by the ability of heat denatured cytosol to inhibit rhURO-D activity. Mice heterozygous for a targeted disruption at the URO-D locus (URO-D+/-) exhibited half the URO-D activity of homozygous controls prior to treatment. After treatment, these animals showed URO-D activity and rhURO-D inhibitory activity comparable to similarly treated wild type (URO-D +/+) mice but with significantly greater uroporphyrin accumulation. With only 10 days of treatment, URO-D +/- but not URO-D +/+ mice showed changes similar in magnitude to those seen after 21 days. Prior to treatment, URO-D genotype did not influence overall hepatic P450 concentration in either sex and there was no significant difference between sexes. The treatment regimen significantly elevated P450 in animals of either URO-D genotype and in both sexes, although the induction response at the 10-day point was attenuated in URO-D +/- mice. From differences in the CO absorbance maximum, and by P450 activity analysis, this attenuated induction response resulted from an attenuation of the CYP2B not the CYP1A induction.

CYP3A-inducing agents and the attenuation of uroporphyrin accumulation and excretion in a rat model of porphyria cutanea tarda.

An experimental model of porphyria cutanea tarda (PCT) can be achieved in 3 weeks by a single injection of a mixture of polychlorinated biphenyls (Aroclor 1254) into iron-loaded female Fischer 344 rats maintained continuously on delta-aminolevulinic acid-supplemented drinking water. In this model, daily treatment with 5-pregnen-3 beta-ol-20-one-16 alpha-carbonitrile (pregnenolone 16 alpha-carbonitrile) attenuated uroporphyrin and heptacarboxylporphyrin accumulation and excretion by 75%. Pregnenolone 16 alpha-carbonitrile treatment had only a minor effect on hepatic iron stores, and it had no effect on the induction of CYP1A activities by Aroclor 1254. In the absence of Aroclor 1254, pregnenolone 16 alpha-carbonitrile had no effect on the accumulation and excretion of highly carboxylated porphyrins. Attenuation of porphyrin accumulation could also be demonstrated with daily troleandomycin treatment. Troleandomycin increased CYP3A-dependent erythromycin demethylase activity, but to a lesser extent than pregnenolone 16 alpha-carbonitrile. Much of the CYP3A induced by troleandomycin was sequestered as a catalytically inactive metabolic-intermediate complex. In the absence of Aroclor 1254, troleandomycin had no effect on the accumulation and excretion of highly carboxylated porphyrins, nor did troleandomycin alter the induction of CYP1A by Aroclor 1254. The results suggest that the major attenuation of hepatic accumulation and urinary excretion of uro- and heptacarboxylporphyrins in the rat PCT model by pregnenolone 16 alpha-carbonitrile and troleandomycin is due to an enhancement of CYP3A catalytic activity.

Randomised controlled trial of eutectic mixture of local anaesthetics cream for venepuncture in healthy preterm infants.

AIM: To assess the safety and efficacy of EMLA cream (eutectic mixture of local anaesthetics) used to induce surface anaesthesia for venepuncture in healthy preterm infants. METHODS: Nineteen infants, median gestational age 31 weeks (range 26-33 weeks) were assessed in a randomised, double blind, placebo controlled, cross-over trial. Changes in physiological variables (heart rate, blood pressure, oxygen saturation) and behavioural responses (neonatal facial coding system score, crying time) before and after venepuncture with EMLA cream were compared with those obtained with a placebo cream to assess efficacy. Toxicity was assessed by comparing methaemoglobin concentrations at 1 hour and 8 hours after application. RESULTS: There was no significant difference in efficacy between EMLA and placebo creams in physiological and behavioural responses. There was no significant difference in methaemoglobin concentrations one hour after the cream had been applied. At eight hours, however, concentrations were significantly higher after EMLA than placebo (p = 0.016). There was no evidence of clinical toxicity. CONCLUSION: This study does not support the routine use of EMLA for venepuncture in healthy preterm infants.

Cytochrome P450 induction, uroporphyrinogen decarboxylase depression, porphyrin accumulation and excretion, and gender influence in a 3-week rat model of porphyria cutanea tarda.

An experimental model of porphyria cutanea tarda, consisting of depressed hepatic uroporphyrinogen decarboxylase (URO-D) activity and accumulation of highly carboxylated porphyrins in the liver, was produced in 3 weeks in Fischer 344 rats. A single administration of a polychlorinated biphenyl mixture (Aroclor 1254) to iron-loaded female rats maintained continuously on delta-aminolevulinic acid supplemented drinking water produced the porphyric state. Without iron loading, URO-D activity appeared slightly less inhibited (33% of normal vs 23% of normal) but porphyrin accumulation was dramatically less (70 vs 605 micrograms porphyrin/g liver). Similar treatment in male rats produced URO-D activities of 54 and 70% of normal with and without iron loading, respectively, and porphyrin concentrations of 76 and 17 micrograms/g. When hexachlorobenzene was substituted for Aroclor 1254 treatment in female rats, URO-D activity was 61 and 69% of normal (with and without iron loading, respectively) and liver porphyrin concentrations were 96 and 25 micrograms/g, respectively. Hexachlorobenzene did not produce significant porphyric effects in male rats. Aroclor 1254 induced CYP1A to a greater extent in females than in males and to a greater extent than hexachlorobenzene, which showed a greater propensity to induce CYP2B. Overall correlation between URO-D activity depression and porphyrin accumulation was highest when fitted to an exponential curve, indicating the importance of the extreme of the depression URO-D activity in evoking experimental porphyria cutanea tarda.

Characterization and expression of iron regulatory protein 2 (IRP2). Presence of multiple IRP2 transcripts regulated by intracellular iron levels.

Iron regulatory proteins (IRP1 and IRP2) are RNA-binding proteins that bind to stem-loop structures, termed iron-responsive elements (IREs), present in either the 5'- or 3'-untranslated regions of specific mRNAs. The binding of IRPs to 5'-IREs inhibits translation of mRNA, whereas the binding of IRPs to 3'-IREs stabilizes mRNA. To study the structure and regulation of IRP2, we isolated cDNAs for rat and human IRP2. The derived amino acid sequence of rat IPR2 is 93% identical with that of human IRP2 and is present in lower eukaryotes, indicating that IRP2 is highly conserved. IRP1 and IRP2 share 61% overall amino acid identity. IRP2 is ubiquitously expressed in rat tissues, the highest amounts present in skeletal muscle and heart. IRP2 is encoded by multiple mRNAs of 6.4, 4.0, and 3.7 kilobases. The 3'-untranslated region of rat IRP2 contains multiple polyadenylation signals, two of which could account for the 4.0-kb and 3.7-kb mRNAs. The 3.7-kb mRNA is increased in iron-depleted cells and occurs with a reciprocal decrease in the 6.4-kb transcript. These data suggest that the 3.7-kb mRNA is produced by alternative poly(A) site utilization in iron-depleted cells.

Effects of chronic corticosteroids and vitamin A on the healing of intestinal anastomoses.

The ability of vitamin A to reverse the inhibitory effects of chronic corticosteroids on cutaneous and fascial wound healing is well established. To investigate this in the unique low-collagen environment of the intestinal anastomosis, 35 rabbits received twice-daily injections of either saline (control), dexamethasone (0.1 mg/kg/day), dexamethasone plus low-dose vitamin A (1,000 IU/kg/day), or dexamethasone plus high-dose vitamin A (10,000 IU/kg/day) for a 2-week period. Animals then underwent creation of single-layer, inverting small and large intestine anastomoses. All injections were continued postoperatively. A fifth group received only dexamethasone preoperatively and dexamethasone plus high-dose vitamin A postoperatively. On postoperative day 7, animals underwent in situ assessment of anastomotic bursting pressure and subsequent histologic examination using a modified Ehrlich/Hunt scale. Corticosteroids significantly impaired the healing of small and large intestine anastomoses, with decreased bursting pressures and histologic parameters at 1 week. Only high-dose vitamin A significantly reversed this inhibitory effect, whether given preoperatively or only postoperatively.

Acute ileus from steroid withdrawal simulating intestinal obstruction after surgery for ulcerative colitis.

Sixty of 127 prednisone-dependent patients with ulcerative colitis who underwent colectomy and endorectal ileal pull-through with ileal reservoir and subsequent laparotomy with ileostomy closure (254 operations) during a 4-year period developed 95 episodes of intestinal obstruction during the early post-operative period. Acute ileus due to steroid withdrawal caused symptoms of intestinal obstruction in 43 patients (76 episodes), whereas true mechanical small-bowel obstruction occurred in only 17 patients (19 episodes). Symptoms of both conditions were similar; however, hypoactive bowel sounds, acute onset of emotional depression, no evidence of obstruction on radiologic contrast stomatogram or enema, and prompt relief of symptoms within 4 hours after intravenous administration of hydrocortisone acetate distinguished acute steroid withdrawal. Since ileus from acute steroid withdrawal occurred four times as frequently as mechanical small-bowel obstruction, prompt recognition and treatment should appreciably reduce postoperative morbidity and hospital costs.

Use of a personal microcomputer for orthoptic therapy.

Personal microcomputers have become almost commonplace in the last few years. Their great flexibility and ability to present graphic images on demand would seem to make them ideal tools for use in a visual training program. We have written such a program for doing fusional vergence therapy that interacts with the patient and stores the results of the training sessions. The features of the program are described and the results from two patients are summarized.