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Objectives: Acute intermittent porphyria (AIP) is caused by mutations in the porphobilinogen deaminase (PBGD) gene that disrupt the function of the enzyme. Many mutations that lead to decreased PBGD activity have been described. An Arg to Trp substitution at codon 173 (CGG→TGG in exon 10) and designated R173W, which leads to a CRIM-negative phenotype, has been reported in Swedish, Finnish, Scottish, and South African kindreds, and in a Nova Scotian proband with fatal AIP. In this work, we investigated the presence of this mutation in a Nova Scotian patient population presenting with AIP. Design and Methods: Single-strand conformation polymorphism analysis and DNA sequencing by TA cloning and Sanger’s dideoxy chain termination method, were used to confirm the maternal transmission of this mutation to the proband. The mutation also eliminates an NciI (also MspI) endonuclease restriction site, which allows for detection of the mutant allele by polymerase chain reaction amplification and restriction enzyme digestion. Results: The family of the Nova Scotian proband and four other AIP kindreds showed the presence of the same mutation. These five families are descendants of German, Swiss, and French immigrants historically known as the “Foreign Protestants,” who were recruited to Nova Scotia in the 1750s. Conclusion: In all these families, descent from one couple that settled in Nova Scotia in 1751 has been identified by genealogy research, consistent with a founder effect within this population. This is the first identified mutation in PBGD causing AIP that has been linked to a founder effect in descendants of an immigrant population to North America, and which could be traced to such a distant background, similar to the South African variegate porphyria mutation.

At the present time, the use of molecular techniques for diagnosing the neurologic porphyrias is limited mainly to those laboratories doing research in the area. Because any of four genes may be involved, it is important to confirm the diagnosis in the index case before undertaking molecular investigation. With advances in methodology, it may eventually become possible to test for the many mutations that cause these disorders in a simple, cost-effective manner.

Acute intermittent porphyria is a neurologic disorder caused by a partial deficiency of porphobilinogen (PBG) deaminase, the third enzyme in the synthetic pathway for heme. The isolation and characterization of the gene for PBG deaminase has brought molecular techniques for diagnosing the disease within reach. Over 60 mutations causing acute intermittent porphyria have been found, most of which are confined to one or several families. Because no single mutation accounts for more than a fraction of cases, screening techniques for locating and identifying unknown mutations are very important. Once a mutation has been characterized, testing of family members is straightforward, and gene carriers can be identified or excluded with greater accuracy than is possible with conventional biochemical tests.

We describe a fluorometric method for screening and quantifying porphyrins in stool. A small sample of stool is extracted with concentrated HCI and is diluted 200-fold in 3 mol/L HCI before analysis. An excitation scan is done from 350 to 450 nm, monitoring emission at 603 nm. Total porphyrin is estimated at the isosbestic point for coproporphyrin and protoporphyrin (402.5 nm). Monitoring emission at 603 nm eliminates interference from chlorophyll, obviating the need for extraction with ether. The position of the excitation peak gives some indication of the nature of the porphyrins in the stool. The acid extract can be injected directly into an HPLC system for fractionation studies. Our method correlates well with the spectrophotometric method developed by Lockwood et al. (Clin Chem 1985;31:1163-7). However, in our method, the sample is easier to process and the assay has higher sensitivity than their assay. The reference interval for porphyrin in healthy individuals by the fluorometric method is less than 300 nmol/g dry weight. We can detect as little as 1 nmol of porphyrin per gram (dry weight) of stool. Results of the method vary linearly with stool porphyrin concentrations as great as 4000 nmol/g dry weight. The within-run imprecision of the method is 3%.

The present study provides a preliminary evaluation of mesoscale circulations forced by surface gradients of heating arising from irrigated areas adjacent to dry land, utilizing a combination of satellite, observational, and modeling approaches. The irrigated crop areas of northeast Colorado were chosen for the study. For the cases studied satellite surface infrared temperature data indicated a typical temperature contrast of approximately 10 K at noon, between the irrigated area and the adjacent dry land. Surface observations and aircraft measurements within the lower region of the atmospheric boundary layer indicated, in general, a significant temperature contrast and moisture difference, thereby implying a potential thermally driven circulation. The anticipated thermally induced flows, however, were reflected in the measurements only by modest changes in the wind speed and wind direction across the contrast location. It is suggested that the daytime, elevated, terrain-forced flow in the area, ...

Information on changes in the urinary excretion pattern of porphyrins can be especially useful in the diagnosis of disorders of porphyrin metabolism. Most clinical laboratory procedures are designed for assay of uroporphyrin and coproporphyrin only, and in many cases even these are not cleanly separated. Hence, we developed a "high-performance" liquid-chromatographic procedure to separate and quantify all five urinary porphyrins--that is, those with four through eight carboxyl groups. Before chromatography, the porphyrins are isolated from other urinary components by two simple, rapid pretreatment steps, then injected into the chromatograph in nonesterified form. They are separated and eluted with a step gradient of methanol/phosphate buffer, pH 3.0, in which the methanol content is first 650, then 850 mL/L. As little as 1 ng of eluted porphyrins can be measured fluorometrically. Analytical recovery of coproporphyrin is virtually 100% and of uroporphyrin 75-80%. CVs are about 10% for coproporphyrin at 70 micrograms/L and 20-40% for uroporphyrin at 8 micrograms/L.

With this electrophoretic method the liver, biliary, and bone isoenzymes of alkaline phosphatase are clearly separated on agarose gels. Wheat-germ lectin, incorporated in the gel matrix, binds the bone isoenzyme selectively, forming a precipitate near the origin. Neither liver nor biliary isoenzyme is affected. Activity staining with an indigogenic dye substrate reveals the liver isoenzyme migrating nearest the anode, followed by the biliary and bone isoenzymes. Results are generally similar to those of electrophoresis on cellulose acetate. However, the lectin-agarose gels better resolve the liver and bone isoenzymes, and heat treatment of samples is not required before electrophoresis.

Most techniques of clot detection do not give information about the mechanical properties of the forming clot. A procedure was developed by which the dynamic loss modulus, or mechanical impedance, of a solution of fibrinogen could be continuously monitored during clot development. This method was then applied to the analysis of purified preparations of normal fibrinogen and two dysfibrinogens. Both thrombin and reptilase were used as clotting agents, and unique tracings of clot impedance vs. time were generated for each dysfibrinogen. The functional defect was known in each case, and the clot impedance tracings were able to distinguish between abnormalities in the release of fibrinopeptides A and B. Mechanical impedance measurements were shown to complement other types of analyses now used in the characterization of dysfibrinogens. In particular, they distinguished between dysfibrinogens that appeared similar by more conventional laboratory methods.

A Ca2+-dependent regulator protein of cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.17) has previously been isolated from rat testis and shown to be a heat-stable, Ca2+-binding protein with a molecular weight of approximately 17,000. The Ca2+-dependent regulator protein is also structurally similar to troponin-C, the Ca2+-binding component of muscle troponin and Ca2+ mediator of muscle contraction. The present report describes a partial amino acid sequence of the Ca2+-dependent regulator. The protein (148 amino acids) is 50% homologous with skeletal muscle troponin-C, but is 11 residues shorter than the muscle protein. The Ca2+-dependent regulator protein has an NH2-terminal sequence of acetyl-Ala-Asp-Glu, a COOH-terminal sequence of Thr-Ala-Lys and 1 residue of epsilon-trimethyllysine located at position 115. All of these properties are distinct from those of other homologous Ca2+-binding proteins. These properties may account for the biological specificities demonstrated by these proteins as compared to the Ca2+-dependent regulator protein. Based on the sequence and a comparison of the Ca2+-dependent regulator protein to other calcium-binding proteins, our data support the view that all of these moecules contain common sequences, especially at their proposed metal-binding sites.

We investigated the composition of the precipitate that forms when wheat-germ lectin derived from Triticum vulgaris is added to serum. A number of serum proteins are precipitated, representing about 2.5% of the total serum protein. This study demonstrates that the interaction of this lectin with the bone isoenzyme of alkaline phosphatase is not specific.

With this electrophoretic method the liver, biliary, and bone isoenzymes of alkaline phosphatase are clearly separated on agarose gels. Wheat-germ lectin, incorporated in the gel matrix, binds the bone isoenzyme selectively, forming a precipitate near the origin. Neither liver nor biliary isoenzyme is affected. Activity staining with an indigogenic dye substrate reveals the liver isoenzyme migrating nearest the anode, followed by the biliary and bone isoenzymes. Results are generally similar to those of electrophoresis on cellulose acetate. However, the lectin-agarose gels better resolve the liver and bone isoenzymes, and heat treatment of samples is not required before electrophoresis.

We evaluated a silver-binding assay for use in measuring total protein in cerebrospinal fluid. The advantage of this procedure over other methods is that, because of its sensitivity, it requires only a 0.5-microL sample. The procedure, which takes approximately 40 min to complete, involves dilution of 0.5-microL samples to 1 mL with distilled water containing sodium dodecyl sulfate, followed by addition of glutaraldehyde and an ammoniacal silver solution. After color development for 30 min, the reaction is terminated with sodium thiosulfate and the absorbance is measured at 420 nm. This assay displayed within-run and day-to-day precision (CV) of 3.1% to 13% over the range of 210 to 1370 mg/L. It showed substantially less protein-to-protein variation than the Coomassie Blue dye-binding procedure when tested with albumin, globulin, and transferrin. It also yielded an accurate estimation of hemoglobin. Moreover, preliminary studies suggested that it was capable of quantifying immunoglobulin light chains and glycoproteins. In a study of 54 human cerebrospinal fluid samples, results of the silver-binding assay corresponded more closely with those obtained with a rate biuret assay (intraclass correlation coefficient = 0.91) than did either the dye-binding or classical Lowry methods.

We investigated the composition of the precipitate that forms when wheat-germ lectin derived from Triticum vulgaris is added to serum. A number of serum proteins are precipitated, representing about 2.5% of the total serum protein. This study demonstrates that the interaction of this lectin with the bone isoenzyme of alkaline phosphatase is not specific.