The uses and properties of PEG-linked proteins. Poly(ethylene glycol) (PEG) is a water soluble polymer that when covalently linked to proteins, alters their properties in ways that extend their potential uses. PEG-modified conjugates are being exploited in many different fields. The improved pharmacological performance of PEG-proteins when compared with their unmodified counterparts prompted the development of this type of conjugate as a therapeutic agent. Enzyme deficiencies for which therapy with the native enzyme was inefficient (due to rapid clearance and/or immunological reactions) can now be treated with equivalent PEG-enzymes. PEG-adenosine deaminase has already obtained FDA approval. PEG-modified cytokines have been constructed and, interestingly, one of the conjugates, PEG-modified granulocyte-macrophage colony-stimulating factor, showed dissociation of two biological properties. This novel observation may open new horizons to the application of PEGylation technology. The biotechnology industry has also found PEG-proteins very useful because PEG-enzymes can act as catalysts in organic solvents, thereby opening the possibility of producing desired stereoisomers, as opposed to the racemic mixture usually obtained in classical organic synthesis. Covalent attachment of PEG to proteins requires activation of the hydroxyl terminal group of the polymer with a suitable leaving group that can be displaced by nucleophilic attack of the epsilon-amino terminal of lysine residues (other nucleophilic groups can also interact). Several chemical groups have been exploited to activate PEG, thereby giving rise to a variety of PEG-proteins. Some of these varieties retain part of the activating group as a coupling moiety between PEG and protein and others provide a direct linkage. For each particular application, different coupling methods provide distinct advantages.(ABSTRACT TRUNCATED AT 250 WORDS)


Skin absorption enhancers.

Skin absorption enhancers. When we try to maximize drug flux through the skin, we usually meet major difficulties because of the impervious nature of the stratum corneum. A popular solution incorporates penetration enhancers into transdermal products. Such materials ideally possess the sole property of reversibly reducing the barrier resistance of the horny layer, allowing the drug to reach the living tissues at a greater rate. This article considers examples of accelerant action that support a general concept explaining enhancer activity in human skin. The core of the proposal is that enhancers usually work by one or more of three main mechanisms: alteration of the lipid or protein domains of the stratum corneum or increase in tissue partitioning of a drug, a coenhancer, water, or any combination of these three chemicals. We may usefully refer to the overall hypothesis as the lipid-protein-partitioning (LPP) concept.


DNA-directed aniline mustards based on 9-aminoacridine: interaction with DNA. A series of 4-substituted aniline mustards ArNH(CH2)nOpC6H4N(CH2CH2Cl)2, where Ar is an acridine and n varies from 2 to 5, interact with DNA. Scatchard analysis shows the compounds bind tightly, with a binding site size similar to that of 9-aminoacridine. The rate of hydrolysis of the mustards, measured by HPLC, is essentially constant across the series. With increasing length of the polymethylene linker, non-covalent binding becomes less strong, but the rate of DNA alkylation increases. Viscometric helix extension measurements and electrophoretic analyses using closed circular supercoiled DNA show that all the compounds are DNA intercalating ligands. Despite these similarities, the compounds are known to have quite different patterns of DNA alkylation, switching from guanine to adenine alkylation as the chain length is extended.


Isotopically sensitive regioselectivity in the oxidative deamination of a homologous series of diamines catalyzed by diamine oxidase. The equivalence of aminomethylene groups in selected diamine substrates of diamine oxidase was exploited for the determination of intramolecular isotope effects. In the series of substrates, [1,1-2H2]-1,3-diaminopropane, [1,1-2H2]-1,5-diaminopentane, [1,1-2H2]-1,6-diaminohexane, [1,1-2H2]-1,7-diaminoheptane and [alpha,alpha-2H2]-4-(aminomethyl)benzylamine, the preference of the enzyme for reaction at the unlabeled methylene was found to vary from 1.45 to 10.5-fold. The observed partitioning ratios go through a minimum value with 1,5-diaminopentane, the best substrate of diamine oxidase of the compounds tested. The results suggest that fast substrates have less opportunity to reorient into alternate binding conformations while bound to the active site of the enzyme. On the other hand, diamine substrates tested that cannot exist in energetically favorable conformations with internitrogen distances of about 7-8 A showed larger intramolecular isotope effects.


Sphingosine induced release of K+ and 5-HT in platelets should not be confused with membrane leakiness. The release of 43K+, lactate dehydrogenase (LDH) and [14C]-5-hydroxytryptamine ([14C]-5-HT) from platelets treated with sphingosine and four differently charged model amphiphiles was studied. Sphingosine was found to differ from the detergents because it induced a concentration-dependent release of both 43K+ and [14C]-5-HT without causing a release of LDH. The release of [14C]-5-HT preceded the release of 43K+ and it is concluded that these effects are associated with platelet activation. The detergents caused a release of 43K+ followed by a release of LDH without causing a release of [14C]-5-HT. These effects are attributed to a non-specific perturbation of the platelet plasma membrane.


The involvement of selenium in peroxisome proliferation caused by dietary administration of clofibrate to rats. The effects of dietary treatment with clofibrate (0.5% w/w for 10 days) on the livers of selenium-deficient male rats were examined. The peroxisome proliferation (as determined by electron microscopy) in the livers of selenium-deficient animals was much less pronounced than in the case of selenium-adequate rats and no increase in peroxisomal fatty acid beta-oxidation (assayed both as antimycin-insensitive palmitoyl-CoA oxidation and lauroyl-CoA oxidase activity) was observed in the deficient animals. On the other hand, in selenium-deficient rats clofibrate caused increases in the specific activity of microsomal lauric acid omega- and omega-1-hydroxylation and an apparent change in mitochondrial size, seen as a redistribution of mitochondria from the 600 x g(av) pellet to the 10,000 x g(av) pellet, which were approximately 50% as great as the corresponding effects on control animals. Obviously, then, these three different effects of clofibrate are not strictly coupled and may involve at least partially distinct underlying mechanisms. Initial experiments demonstrated that peroxisome proliferation could be obtained by exposing primary hepatocyte cultures derived from selenium-deficient rats to clofibric acid (an in vivo hydrolysis product of clofibrate which is the proximate peroxisome proliferator), nafenopin or mono(2-ethylhexyl)phthalate. This finding suggests that selenium deficiency does not have a direct influence on the basic process(es) underlying peroxisome proliferation, but rather has indirect effects, influencing, for example, the pharmacokinetics of clofibrate and/or hormonal factors.


The mechanism of Hg2+ toxicity in cultured human oral fibroblasts: the involvement of cellular thiols. To study amalgam-related toxicity in a primary target cell type, human oral fibroblasts were grown in a low-serum medium containing 1.25% fetal bovine serum and exposed to Hg2+, a corrosion product of amalgam. A 1-h exposure to various concentrations of Hg2+ resulted in a dose-dependent loss of colony forming efficiency. Removal of the low-molecular-weight thiol cysteine from the medium increased the toxicity of Hg2+ almost 50-fold in comparison with complete medium or medium without fetal bovine serum. Accordingly, fetal bovine serum was not found to contain detectable levels of low-molecular-weight thiols. The levels of cellular free protein thiols were shown to be depleted Hg2+ at significantly lower concentrations of the metal ion than those required to decrease the levels of the major cellular low-molecular weight thiol glutathione. These decreases were dependent on the exposure conditions, i.e. the presence of serum and thiols, in a manner similar to the effect on colony forming efficiency. Other functions commonly related to cell viability, including the accumulation of the vital dye neutral red, the cytosolic retention of deoxyglucose and the mitochondrial reduction of tetrazolium were also inhibited by Hg2+, albeit at higher concentrations. Finally, the depletion of cellular glutathione, by pre-exposure of the cells to the glutathione synthesis inhibitor buthionine sulfoximine, somewhat increased the toxicity of Hg2+ and potentiated the depletion of protein thiols. Taken together, the toxicity of Hg2+ in human oral fibroblasts was demonstrated in several assays of which colony forming efficiency was the most sensitive, cell killing by this agent was related to its high affinity for protein thiols, whereas glutathione showed a significant, but limited, ability to protect the cells from Hg2+ toxicity.


Ultrasonic diagnosis of oral and neck malignant lymphoma. A series of 14 patients with nodal and extranodal non-Hodgkin's lymphoma of the oral and neck region was analyzed by ultrasonogram evaluation. Eight nodal lymphomas and six extranodal lymphomas commonly exhibited almost completely similar ultrasonographic findings, specifically, clear delineation of the boundary echo and a homogeneous, weak internal echo, the so-called pseudo-liquid-like images. The results derived from our study suggest that ultrasonic diagnosis is also helpful in evaluating patients with lymphoma during the initial diagnosis and initial treatment like other diagnostic imaging modalities.


Cardiotoxicity of mitomycin A, mitomycin C, and seven N7 analogs in vitro. The alkylating antitumor agents mitomycin A (MMA), mitomycin C (MMC), and seven N7 analogs were compared in terms of their cardiotoxic and antitumor activity in vitro. Neonatal rat-heart myocytes were sensitive to five of the compounds studied, including MMA, 7-dimethylamidinomitosane (BMY-25282), 7-(N-methyl-piperazinyl)-mitosane (RR-194), N7-(4-iodophenyl)-MMC (RR-208), and N7-(4-hydroxyphenyl)-MMC (M-83) in order of descending molar potency. MMA and RR-208 possessed the greatest cytotoxic potency against 8226 human myeloma tumor cells in vitro. Two of the nine mitomycins studied, BMY-25282 and M-83, showed greater cytotoxic potency for heart cells. For these two agents, the ratio of the 50% inhibitory concentration in heart cells to that in 8226 myeloma cells was 50 and 32, respectively. For the other analogs, the tumor-cell cytotoxic potency was much higher (ranging from 200 to 7,000). For the nine mitomycin compounds, a correlation was found between heart-cell toxicity and low reduction potentials (E1/2 values) ranging from -0.16 to -0.37 V. Thus, as the reduction potential decreased (easier reducibility), the cardiotoxic potency in vitro increased (r = 0.81). In contrast, mitomycins with reduction potentials of higher than -0.37 V were much less potent cardiotoxins. Thus, mitomycin C (E1/2 = -0.45 V) was noncardiotoxic even when tested at concentrations 100-fold above those pharmacologically achievable in humans. Mitomycin C also failed to enhance doxorubicin (Adriamycin) cardiotoxicity in vitro. Importantly, no correlation was found between the reduction potential and the antitumor activity of the nine analogs (n = 0.51), in this small series.


Cell cycle-specific metabolism of arabinosyl nucleosides in K562 human leukemia cells. Exponentially growing K562 cells incubated with 1-beta-D-arabinofuranosylcytosine (ara-C) accumulate ara-C triphosphate (ara-CTP) at a higher rate and to a greater concentration after pretreatment with 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) than do cells treated with ara-C alone. Potentiation of ara-C metabolism is due in part to an indirect effect of F-ara-A triphosphate (F-ara-ATP)-mediated reduction in deoxynucleotide pools and consequent activation of deoxycytidine kinase. Because the levels of deoxynucleotide pools and the activity of deoxycytidine kinase are cell cycle-specific, we investigated the effect of cell cycle phases on the accumulation of ara-CTP and the influence of F-ara-A pretreatment on such accumulation. Exponentially growing K562 cells were fractionated into G1, S, and G2+M phase-enriched subpopulations (each enriched by > 60%) by centrifugal elutriation. The rate of ara-CTP accumulation was 22, 25, and 14 microM/h and the rate of F-ara-ATP accumulation was 38, 47, and 33 microM/h in the G1, S, and G2+M subpopulations, respectively. The rate of elimination of arabinosyl triphosphates was similar among the different phases of the cell cycle. After pretreatment with F-ara-A, the rate of ara-CTP accumulation in the G1, S, and G2+M phase-enriched subpopulations was 43, 37, and 26 microM/h, indicating a 1.7-, 1.5-, and 1.9-fold increase, respectively. These results suggest that a combination of F-ara-A and ara-C may effectively potentiate ara-CTP accumulation in all phases of the cell cycle. This observation is consistent with the results of studies on the modulation of ara-C metabolism by F-ara-A in lymphocytes and leukemia blasts obtained from patients with chronic lymphocytic leukemia and acute myelogenous leukemia, respectively.


Distribution of hexadecylphosphocholine and octadecyl-methyl-glycero-3-phosphocholine in rat tissues during steady-state treatment. The distribution of the alkylphosphocholine hexadecylphosphocholine (He-PC) and the (alkyl)lysophospholipid 1-0-octadecyl-2-0-methyl-rac-glycero-3-phosphocholine (ET18-OCH3) was analyzed in rats. The compounds were given orally at a daily dose of 75 mumol/kg body weight. After 6, 11, and 18 days, three rats in each treatment group were killed and the drug concentration in various tissues and fluids was determined. With the exception of the kidney (He-PC) and brain (He-PC and ET18-OCH3), steady-state levels of the drugs could be achieved in all organs investigated and in serum. Maximal concentrations of He-PC were found in the kidney, adrenal glands, and spleen, whereas the highest concentrations of ET18-OCH3 were detected in the adrenal glands, spleen, and small intestine. The concentrations of He-PC exceeded those of ET18-OCH3 in most tissues by a factor of about 2-25. Since samples of urine and feces did not contain detectable amounts of the compounds, the absorption of both lipid analogues was assumed to be complete. The total amount of He-PC recovered after 6, 11, and 18 days was 15%, 12%, and 6%, respectively, and that of ET18-OCH3 was 1.3%, 0.8%, and 0.3%, respectively. This indicates that the bioavailability of He-PC and ET18-OCH3 is not controlled by differences in the uptake of the two drugs, but by differences in their metabolism. The results could explain the differing efficacy of these two compounds in their antitumor action in animal models.


The role of NAD(P)H:quinone oxidoreductase in mitomycin C- and porfiromycin-resistant HCT 116 human colon-cancer cells. A mitomycin C (MMC)- and porfiromycin (PFM)-resistant subline of the HCT 116 human colon-cancer cell line was isolated after repeated exposure of HCT 116 cells to increasing concentrations of MMC under aerobic conditions. The MMC-resistant subline (designated HCT 116-R30A) was 5 times more resistant than the parent cells to MMC and PFM under aerobic conditions. Both the MMC-resistant cells and the parent HCT 116 cells accumulated similar amounts of PFM by passive diffusion, but levels of macromolecule-bound PFM were about 50% lower in the resistant cell line, implying a decrease in PFM reductive activation in the resistant cells. The finding that microsomes from either sensitive or resistant cells showed an equal ability to reduce MMC and PFM indicated that the activity of NADPH cytochrome P-450 reductase (EC 1.6.2.4) was not changed in the resistant subline. Soluble extracts of HCT 116 cells reduced MMC and PFM more effectively at pH 6.1, and NADH and NADPH were utilized equally well as electron donors under both aerobic and anaerobic conditions. These data suggest that quinone reductase (EC 1.6.99.2; DT-diaphorase) in soluble extracts is responsible for the reduction of MMC. Quinone reductase activities in soluble extracts of HCT 116-R30A cells for the reduction of dichlorophenol indophenol (DCPIP) and menadione-cytochrome c at optimal pHs were decreased by 95% as compared with those obtained in parent cells. However, the MMC-reducing activity of HCT 116-R30A soluble extracts was only 50% lower than that of the parent cell extracts. The kinetic constants (Km, Vmax) found for quinone reductase in the two cell lines with respect to the substrates DCPIP and menadione differed. Two species of mRNA for quinone reductase (2.7 and 1.2 kb) were detected in both cell lines, and there was no detectable difference between parent and resistant cells in the steady-state level of either of these mRNA species. Furthermore, incubation with the quinone reductase inhibitor dicoumarol rendered HCT 116 cells more resistant to MMC. Alteration of the quinone reductase activity in HCT 116-R30A cells appears to be the mechanism responsible for their resistance to MMC and PFM.


Intraperitoneal administration of the antitumour agent N-[2-(dimethylamino)ethyl]acridine-4-carboxamide in the mouse: bioavailability, pharmacokinetics and toxicity after a single dose. The pharmacokinetics, tissue distribution and toxicity of the antitumour agent N-[2-(dimethylamino)-ethyl]acridine-4-carboxamide (AC) were studied after i.p. administration of [3H]-AC (410 mumol/kg) to mice. The latter is the optimal single dose for the cure of advanced Lewis lung tumours. AC was rapidly absorbed into the systemic circulation after i.p. administration, with the maximal concentration (Cmax) occurring at the first time point (5 min). There was no reduction in bioavailability as compared with previous i.v. studies, but the shape of the plasma concentration-time profile was considerably different, reflecting a 3-fold lower Cmax value (20.9 +/- 3.6 mumol/l) and a longer t1/2 value (2.7 +/- 0.3 h) as compared with that observed after i.v. administration (1.6 +/- 0.6 h). Model independent pharmacokinetic parameters after i.p. administration were: clearance (C), 17.5 l h-1 kg-1; steady-state volume of distribution (Vss), 14.1 l/kg; and mean residence time (MRT), 1.46 h. High but variable tissue uptake of AC was observed, with tissue/plasma AUC ratios being 5.7 for heart, 8.4 for brain, 18.9 for kidney and 21.0 for liver but with similar elimination t1/2 values ranging from 1.3 to 2.7 h. All radioactivity profiles in plasma and tissues were greater than the respective parent AC profiles and showed prolonged elimination t1/2 values ranging from 21 h in liver to 93 h in brain. However, tissue/plasma radioactivity AUC ratios were near unity, ranging from 0.7 to 1.57, with the exception of the gallbladder (15.6), which contained greater amounts of radioactivity. By 48 h, approximately 70% of the total dose had been eliminated, with the faecal to urinary ratio being approximately 2:1. This i.p. dose was well tolerated by mice, with sedation being the only obvious side effect. No major change was observed in blood biochemistry or haematological parameters. Comparisons of Cmax, tmax and AUC values determined for AC in brain after its i.p. and i.v. administration suggest that the reduction in acute toxicity after i.p. administration is not due to reduced exposure of the brain to AC as measured by AUC but may be associated with the lower Cmax value or the slower rate of entry of AC into the brain after i.p. administration.


Cyclosporin A potentiation of VP-16: production of long-term survival in murine acute lymphatic leukemia. Our prior in vitro studies on the correction of multidrug resistance by cyclosporin A (CsA) prompted us to investigate the effect of CsA and VP-16 in vivo. CsA given simultaneously at 2 or 10 mg/kg with VP-16 to BDF/1 mice bearing parental drug-sensitive P388 or L1210 lymphatic leukemia produced a 100% increase in survival as compared with VP-16 treatment alone. CsA-containing regimens also promoted 60-day survival in a significant number of P388 or L1210 leukemia-bearing mice as compared with animals receiving VP-16 in the absence of CsA (P < 0.02 and P < 0.001, respectively). CsA enhancement of the survival of mice bearing these lymphatic leukemias is restricted to VP-16, since the addition of CsA to therapeutic agents such as vincristine, daunorubicin, methotrexate, or cisplatin had no effect on survival.


Distribution characteristics of mitoxantrone in a patient undergoing hemodialysis. The pharmacokinetic profile of mitoxantrone in a patient undergoing hemodialysis is described. Significant characteristics of our patient included lymphoma with liver involvement, tumor lysis syndrome, renal and hepatic failure. Combination chemotherapy consisted of mitoxantrone, vincristine, and cyclophosphamide. Mitoxantrone plasma samples were obtained prior to dosing and at 0, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.5, 7.0, and 12 h after the intravenous infusion of a 17-mg dose over 20 min. Serum concentrations were determined by high-performance liquid chromatography. The serum concentration versus time curve was consistent with a three-compartment model. However, rebounds in serum drug concentrations were detected during the last portion of dialysis and after its completion. The gamma elimination half-life could not be determined due to the continued detection of rebounds in drug concentrations throughout the postdialysis sampling period. The alpha and beta distribution phases did not appear to be affected by hemodialysis. The peak mitoxantrone concentration fell within the reported range. Mitoxantrone does not appear to be eliminated by hemodialysis, and dose adjustments are not needed in patients undergoing this procedure.


Relationship between tumor cell density and drug concentration and the cytotoxic effects of doxorubicin or vincristine: mechanism of inoculum effects. When tumor cell density increases, the cytotoxic activity of certain anticancer agents, such as vincristine (VCR) and doxorubicin (DXR), progressively decreases. This phenomenon is termed the inoculum effect. Since VCR and DXR are less active in an acidic environment, we questioned whether the inoculum effects could have resulted from acidification of the medium that may have developed due to the high cell density. However, measurements of the cytotoxic activity of these agents in a pH-controlled medium revealed only a minor correction of the inoculum effects. Second, we wondered whether the inoculum effects that occurred at the high cell density might have been attributable to insufficient amounts of drugs to bind all the binding sites of the cells. To test this hypothesis, we used drug-resistant sublines, which required higher VCR or DXR concentrations for cell killing than did the parent cell line. When higher drug concentrations were used, the dose-response curves generated for low- and high-density cell populations became closer and overlapped each other, resulting in virtual disappearance of the inoculum effects. Measurements of cellular drug levels revealed that at a high cell density, cells accumulated much smaller amounts of both VCR and DXR in parallel with the positive inoculum effect. In contrast, when high concentrations of the drugs were used in drug-resistant cells, differences in the cellular drug contents between low and high cell densities became narrow. Cisplatin (DDP) belongs to a group of drugs that do not produce inoculum effects, and DDP's cytotoxic effects were not influenced by the pH-controlled medium or by the use of drug-resistant cell lines. These observations indicate that the inoculum effects are the result of the unavailability of VCR or DXR molecules to all cellular binding sites when cells at high densities are exposed to drugs. The drug concentration relative to cell density was apparently the major determinant for the inoculum effects seen in VCR- or DXR-induced cell killing.


Mechanistic implications of alterations in HL-60 cell nascent DNA after exposure to 1-beta-D-arabinofuranosylcytosine. To improve our understanding of the mechanism of 1-beta-D-arabinofuranosylcytosine (ara-C) incorporation into DNA, we investigated the physical properties (size, position of nucleoside incorporation) of small fragments of nascent DNA (nDNA) obtained by pH-step alkaline elution of intact HL-60 cells following their exposure to ara-C. In the pH-step alkaline elution procedure, the smallest fragments of nDNA elute at pH 11. Anion-exchange high-performance liquid chromatography (HPLC) of nDNA obtained by 1 h elution at pH 11.0 of lysed HL-60 cells revealed a preponderance of nDNA fragments ranging from 0.5 to 40 kb in control ([3H]-dThd-labeled) cells. Exposure of cells to ara-C (0.8-1 microM) resulted in a loss of the preponderance of radiolabel in fragments of 0.5-40 kb along with redistribution of the radiolabel (from [3H]-dThd or [3H]-ara-C) into smaller nDNA fragments (predominantly < 100 bases in length) as determined by HPLC. We used the ability of pH-step alkaline elution to provide these small nDNA fragments produced by ara-C to investigate the paradoxical behavior of ara-C as a chain terminator in cell-free DNA synthetic systems while being incorporated into an internucleotide position in intact cells. Following the digestion of purified nDNA with micrococcal nuclease and spleen phosphodiesterase II, the proportion of radiolabel in 3'-dNMP (indicating an internucleotide position) or free nucleoside (indicating a chain terminus position) was determined by reverse-phase HPLC. In digests of prelabeled genomic DNA, as expected, > 90% of the radiolabel from [14C]-dThd or [3H]-ara-C was found to exist in an internucleotide position (as determined by co-chromatography with authentic 3'-dTMP or 3'-ara-CMP). In contrast, digests of nDNA that eluted at pH 11.0 revealed a significantly higher proportion of radiolabel in the chain terminus position (29%-35%) when the nDNA was obtained from cells exposed to 1 microM [3H]-ara-C as compared with cells exposed to [3H]-dThd or [3H]-dCyd alone (< 10%). These data obtained from pH-step alkaline elution of intact cells suggest that by causing the inhibition of chain elongation while failing to inhibit the formation of new nDNA replication intermediates, ara-C exposure leads to the production of very small nDNA fragments. This relative chain-terminating effect of ara-C is most apparent in the small nDNA replication fragments that elute at pH 11.0.(ABSTRACT TRUNCATED AT 400 WORDS)


Protection against fludarabine neurotoxicity in leukemic mice by the nucleoside transport inhibitor nitrobenzylthioinosine. Fludarabine phosphate (F-ara-AMP, Fludara) is rapidly converted in the circulation to fludarabine (F-ara-A) and is among the most effective single agents in the treatment of chronic lymphocytic leukemia. Although current treatment protocols are well tolerated, severe neurotoxicity was a consequence of high-dose F-ara-AMP regimens used in early phase I trials against adult acute leukemia. The present study showed that in mice implanted with leukemia L1210, fatal neurotoxicity, which initially manifested as hind-limb paralysis, was a consequence of high-dose F-ara-AMP treatment. However, the incidence of neurotoxicity was reduced by the coadministration of NBMPR-P, the 5'-phosphate of nitrobenzylthioinosine, a potent inhibitor of the es equilibrative nucleoside transport (NT) system. NBTGR-P, the 5'-phosphate of nitrobenzylthioguanosine (also a potent NT inhibitor) similarly prevented F-ara-AMP neurotoxicity in this experimental system. Treatment with F-ara-AMP/NBMPR-P combinations was more effective with respect to the fractional yield of "cured" mice than were the same treatment regimens without NBMPR-P.


Serum elimination half-life of tamoxifen and its metabolites in patients with advanced breast cancer. In breast cancer patients discontinuing chronic tamoxifen therapy, the serum elimination of metabolites X, Y and E paralleled that of tamoxifen, whereas that of metabolite Z did not. The serum elimination of tamoxifen and metabolites X and B was increased by amino-glutethimide treatment, whereas that of metabolites Z, Y, and E was not.


Adriamycin-induced inhibition of arachidonate incorporation into phospholipids of mastocytoma P815 cells. Adriamycin inhibited [1-14C]-arachidonic acid uptake by mastocytoma P815 cells in a dose-dependent manner (5-120 nM). The incorporation of [1-14C]-arachidonic acid into phospholipids was decreased mainly into cardiolipin and phosphatidylcholine.


Primary liver cancer in Hong Kong.

Primary liver cancer in Hong Kong. From 1984 to 1987, we conducted a series of chemotherapy trials to assess their efficacy and impact on survival in patients with inoperable hepatocellular carcinoma. A total of 18 patients were treated with etoposide at a dose of 200 mg/m2 given on days 1-3 every 3 weeks for a maximum of 8 courses. No response was observed in any of the 18 patients studied. A further trial using epirubicin at doses of 40 and 75 mg/m2 again showed no evidence of a tumor response in 14 consecutive patients. The dose of epirubicin was further escalated to 90 mg/m2. Of the 33 patients treated, 1 (3%) achieved a complete response and 2 (6%) showed a partial response. Although the overall median survival amounted to only 72 days, the survival of the three responders was 15, 13, and 12 months, respectively. Therefore, we believe that the tumor response to epirubicin may be dose-dependent. However, the toxicity was also dose-dependent. To improve the therapeutic index, we are currently evaluating the impact of intrahepatic arterial injection of a lipiodol-epirubicin emulsion.


Liver transplantation in Taiwan: the Chang Gung experience. Between March 1984 and February 1991, six orthotopic liver transplantations were performed at the Chang Gung Memorial Hospital in Taiwan. The indications for transplantation were Wilson's disease (5 patients) and biliary atresia (1 patient). Donors and recipients were matched only for size and ABO blood group compatibility, and the recipient operations were performed without the use of a venovenous bypass. Arterial reconstruction was carried out by end-to-end hepatic artery anastomosis (4), thoracic aortic conduit (1), or interposition of an iliac artery graft (1), whereas biliary reconstruction was accomplished by a choledochocholedochostomy using a T-tube stent (4) or a choledochocholedochostomy using an external cholecystostomy without stenting (2). Biliary complications occurred in three patients, and all required additional surgery. The average duration of donor-liver cold ischemia, operating time, and blood loss during surgery were 7 h and 50 min (range, 4.5-9 h), 13.5 h (range, 11.8-17 h), and 4,385 ml (range, 750-12,000 ml), respectively. The immunosuppressive regimens included a cyclosporin-steroid combination (n = 2) and a triple-drug combination (n = 4). All except one of the surviving patients experienced at least one rejection episode that was reversed by a methyl-prednisolone bolus and/or recycle. One patient developed a primary cytomegalovirus (CMV) infection that responded well to Ganciclovir treatment. Two of the patients died, one of injuries sustained in a traffic accident 3 years after transplantation, and the other of massive upper gastrointestinal bleeding. The overall survival value at 3 months was 83%, and the follow-up period ranged from 3 months to 7 years. All of the survivors have achieved complete rehabilitation and currently enjoy an excellent quality of life with normal liver function. Although the present study involved a small number of cases, our results indicate that liver transplantation can be successfully achieved in a high proportion of patients with acceptable morbidity, mortality, and cost in an Asian setting. The extreme shortage of donor organs is currently the most important obstacle limiting the application of liver transplantation in Taiwan.


Management of patients with unresectable liver metastases from colorectal and gastric cancer employing an implantable port system. Between 1985 and 1990, 50 patients with unresectable liver metastases from colorectal cancer and 34 subjects with metastases from gastric cancer were treated by repeated hepatic arterial infusion chemotherapy employing an implantable prot system. A catheter was inserted into the hepatic artery via the left subclavian artery and was connected to the implantable injection port in each patient. 5-Fluorouracil (5-FU) at 330 mg/m2 per week (167 mg/m2 daily given continuously over the initial 3 months for colorectal cancer), Adriamycin (ADR) at 20 mg/m2 every 4 weeks and mitomycin C (MMC) at 2.7 mg/m2 every 2 weeks were given to all 34 patients with gastric cancer and to 31 of the colorectal cancer patients. The remaining 19 patients with colorectal cancer received 5-FU at 1,000 mg/m2 every week. As a rule the treatment was performed on an outpatient basis. The side effects and complications observed included myelosuppression (23%), hepatic arterial occlusion (21%), and gastroduodenal mucositis (12%), although no major toxicity was encountered. The response rate (CR+PR) among the evaluated patients as determined using CT scans was 67% for colorectal cancer and 73% for gastric cancer. The overall median survival was 12 months and 15 months, respectively. Good local control of liver metastases from the colorectal and gastric cancers was achieved by repeated hepatic arterial infusion chemotherapy employing an implantable port system without the need for hospitalization and without producing major toxicity. Thus, the implantable port system is very useful for the management of patients with unresectable liver metastases.


Interlaboratory study of the IFCC method for alanine aminotransferase performed with use of a partly purified reference material. We present the results of a study on performance of a reference material for alanine aminotransferase (ALT, EC 2.6.1.2) and the corresponding IFCC-approved method in an interlaboratory trial involving 13 laboratories. The ALT material was partly purified from pig heart (specific activity, 150 kU/g) and was essentially free of six potentially contaminating enzyme activities, including aspartate aminotransferase (EC 2.6.1.1). The partly purified ALT was lyophilized in a triethanolamine-buffered matrix, pH 6.4, containing bovine serum albumin and saccharose. Under these conditions, the predicted yearly loss of activity was 0.02% at 4 degrees C and < 0.01% at -20 degrees C. The final blank-corrected results of the accepted set of data gave a mean (SD) of 128.5 (5.1) U/L. The among-laboratory SD was 4.6 U/L and the within-laboratory SD was 2.0 U/L. The certified ALT catalytic concentration in the reconstituted material was 129 U/L with a 0.95 confidence interval of +/- 4 U/L.


Affinity binding assay of glycohemoglobin by two-dimensional centrifugation referenced to hemoglobin A1c. We describe an automated assay of glycohemoglobin performed with the Abbott Vision analyzer. The assay is based on batch affinity-extraction with 3-aminophenylboronic acid-derivatized agarose beads. Reagents are packaged in a disposable test pack. Whole-blood specimens are hemolyzed with saponin within a glass capillary tube inserted into the test pack. The sample is automatically diluted with, mixed with, and separated from the solid-phase reagent. Bichromatic absorbance readings are used to calculate the percentage of hemoglobin bound. Based on the linear correlation between affinity-measured glycohemoglobin and HPLC-measured hemoglobin A1c, the percentage of hemoglobin bound is converted to a "standardized %HbA1c" result by use of regression parameters stored during a calibration run. The combination of affinity methodology with standardization by reference to HPLC produces values directly comparable with those obtained by methods specific for HbA1c. The method produces 10 results within 15 min. The assay operates with CVs < 5%, and the results correlate highly with those by ion-exchange and affinity minicolumn methods, and by ion-exchange HPLC.


Analytical and clinical evaluation of creatine kinase MB mass assay by IMx: comparison with MB isoenzyme activity and serum myoglobin for early diagnosis of myocardial infarction. We analytically and clinically evaluated Abbott's IMx assay for creatine kinase (CK) isoenzyme MB (CK-MB) in serum. Over a 1-year period, the method was more specific but less precise than catalytic isoenzyme measurements by electrophoresis or immunoinhibition. Sera from different individuals without electrophoretic evidence of CK-MB but containing macro CK type 1 (n = 20), mitochondrial CK (n = 5), or CK-BB (n = 5) were scored as CK-MB negative by the IMx. Likewise, CK-MB-negative by the sera remained so after addition of purified human CK-MM (< or = 7600 U/L) or CK-BB (< or = 8100 U/L). For 39 patients admitted for suspicion of uncomplicated acute myocardial infarction (precordial pain for < or = 4 h), the diagnostic performance of the IMx CK-MB assay on admission and 4 h later was superior to that of total CK activity and compared well with that of CK-MB activity measured by electrophoresis or immunoinhibition. An admission, myoglobin showed a higher diagnostic sensitivity, specificity, and predictive value than did CK-MB and was the most informative test. Diagnostic performance on admission and 4 h later was further improved by considering positivity for myoglobin and for CK-MB by IMx and for the change in each over the first 4 h of hospitalization as criteria. Twelve hours after admission, diagnostic performance was further improved for all CK and CK-MB methods but began to decline for myoglobin.


Apolipoprotein B quantified by particle-concentration fluorescence immunoassay. We have developed a particle-concentration fluorescence immunoassay (PCFIA) for estimating apolipoprotein (apo) B concentrations in plasma. A two-step antigen-detection system with a polyclonal antibody to apo B bound to carboxyl-polystyrene particles binds the antigen, and a fluorescein-labeled monoclonal antibody detects the bound apo B. Narrow-cut low-density lipoproteins (d = 1.03-1.05 kg/L) were used as the primary standard. The assay compares well with the enzyme-linked immunosorbent assay. The PCFIA gives parallel responses with low-density lipoprotein, very-low-density lipoproteins, and plasma samples, and can be fully automated and completed in 3 h. In a pilot study of patients with diabetes, coronary artery disease (CAD), or both, we found statistically significant differences in apo B concentrations for patients with both CAD and diabetes compared with those for patients with diabetes alone or for control subjects (P < 0.01).


Use of synthetic H disaccharides as acceptors for detecting activities of UDP-GalNAc:Fuc alpha 1-->2Gal beta-R alpha 1-->3-N-acetylgalactosaminyltransferase in plasma samples from blood group A subgroups. Concentrations of blood group A-specified alpha(1-->3)-N-acetylgalactosaminyltransferase (A enzyme) were measured in human plasma of blood groups A1, A2, and A3 by using chemically synthesized H disaccharides and H type 1 and type 2 trisaccharides attached to hydrophobic aglycones as acceptors. When the trisaccharides were used as acceptors, enzyme activities were reduced in samples from A2 and A3. However, the H disaccharides were shown to be good acceptors even for enzymes from A2 and A3, and no significant difference in enzyme concentration was detected in any of the plasma tested.


Creatine kinase MB isoforms in patients with skeletal muscle injury: ramifications for early detection of acute myocardial infarction. We measured total creatine kinase (CK), CK-MB isoenzyme, and the MB isoforms in 202 serum and plasma samples from nine groups of patients and normal individuals: 39 with acute myocardial infarction (MI), divided according to time between the onset of chest pain and blood collection (1-6 h, 7-12 h, and 13-48 h); 26 with chest pain for whom an MI was ruled out, sampled at admission; 17 undergoing bypass surgery or cardiac catheterization, sampled within 6 h after either procedure; 17 with acute skeletal muscle injury, sampled within 8 h after injury; 30 marathon runners immediately after a race; 17 runners and other athletes > 12 h after training or a race; 12 with cerebral injury or seizures, sampled at admission; 8 with closed head injury, sampled at admission; and 38 normal subjects. CK-MB (relative index) and MB isoforms (MB2/MB1) were respectively increased in 15% and 75% of MI patients 1-6 h after onset, 94% and 94% after 7-12 h, and 88% and 8% after 12 h, and in 87% and 82% of cardiac surgery patients. MB isoforms were increased in most patients with acute skeletal muscle trauma and in subjects examined after exercise, but were within normal limits in patients for whom MI was ruled out, patients with cerebral trauma, and normal individuals. The relative index of MB/total CK was normal in essentially all individuals in the last groups, including those with acute skeletal muscle trauma. We concluded that the CK-MB isoform ratio is increased in both acute skeletal muscle injury and MI. The isoform ratio is most useful for distinguishing recent from old (> 12 h) injury.


Quantitative determination of methemoglobin by measuring the solvent-water proton-nuclear magnetic resonance relaxation rate. We report a new method for the quantitative determination of human methemoglobin (metHb) based on the measurement of the solvent-water proton-nuclear magnetic resonance (NMR) relaxation rate R1 [normalized to 1 mmol/L hemoglobin (Hb) concentration]. MetHb (%) is estimated from the linear dependence of R1 on the metHb concentration, taking into account the simple relationship [MetHb] = [(R1 - R1HbO2)/(R1metHb - R1HbO2)].100, where R1HbO2 and R1metHb are values for the solvent-water relaxation rate of standard 1.0 mmol/L solutions of the oxygenated derivative of human hemoglobin (HbO2) and of metHb, respectively. The minimum metHb that may be determined from the analysis of the experimental data is 0.5 +/- 0.4%.


Standardization of glycohemoglobin determinations in the clinical laboratory: three years of experience. Measurement of glycohemoglobin has been recommended for the long-term assessment of glycemic control in diabetic patients. Because different analytical methods measure different glycohemoglobin species, it has been difficult to compare results between laboratories. Here we report 3 years of experience with calibration of an affinity chromatography method for measuring total glycohemoglobin (GHb). Calibration was achieved by including in each assay three hemolysate calibrators for which values for HbA1c and GHb had been determined by repeated analyses by high-performance liquid chromatography (HPLC) and affinity chromatography, respectively. Calibration improved interassay precision (CV = 3.20-7.90% and < 5.0% before and after the introduction of calibration, respectively) and eliminated lot-to-lot variability. In 91 samples, HbA1c was estimated by the calibrated affinity chromatography assay and measured by an ion-exchange HPLC method. Estimated and HPLC-measured HbA1c showed no clinically significant differences during 36 months. The high degree of long-term precision, the disappearance of lot-to-lot variability, and the excellent comparability between analytical methods measuring different species of glycated hemoglobins demonstrate the advantages of calibration.


Discrepancies between sodium concentrations measured by the Kodak Ektachem 700 and by dilutional and direct ion-selective electrode analyzers. We have identified rare (approximately 0.2% of all samples), but clinically significant, discrepancies between serum or plasma sodium concentrations measured with the Kodak Ektachem 700's direct ion-selective electrode (ISE) method and concentrations measured with two other analyzers: the Beckman Synchron CX3's dilutional ISE instrument and the Radiometer KNA2 instrument for sodium-potassium analysis by the direct ISE method. The differences do not appear to be related to any previously identified sources of discrepancy, such as variations in triglycerides, bicarbonate, total protein, albumin, or gamma-globulin, the presence of paraproteins, or interference by benzalkonium chloride from heparinized catheters. They occurred despite the use of Gen 04 reference fluid on the Ektachem. We could not identify any drug or family of drugs that the patients had taken in common and that might influence the results. Until this problem is resolved, Ektachem users should be aware of the potential for discrepancies of > 6 mmol/L in measurements of sodium concentrations.


DNA diagnosis with mutation-specific artificial methylation sites: application to rapid screening of delta F508. Many polymerase chain reaction (PCR)-based methods for diagnosis of minute mutations are suboptimal for automated screening because of their reliance on gel electrophoresis or probe hybridization. In the method reported here, PCR products containing artificial methylation sites are analyzed by measuring incorporation of radiolabeled methyl groups. Primers are designed to amplify the possible mutation-containing region such that the 3' end of one primer lies adjacent to the possible mutation. Sequence modification near that end creates either a mutation- or wild type (WT)-specific artificial methylation site in the PCR product. The product is briefly incubated with an appropriate DNA methylase and tritiated S-adenosylmethionine ([3H]SAM), separated from free SAM by column chromatography, and analyzed for incorporation of tritium. Applying this technique to the cystic fibrosis delta F508 deletion, we accurately diagnosed five homozygotes, five heterozygotes, and five normal individuals within 40 min of PCR completion. The method can be generalized to rapid, automated detection of a variety of point mutations and small deletions.


Fluorescence polarization immunoassay and HPLC assays compared for measuring monoethylglycinexylidide in liver-transplant patients. We compared fluorescence polarization immunoassay (FPIA, x) and HPLC (y) for measuring monoethylglycinexylidide (MEGX) concentrations in 119 serum samples from 61 liver-transplant donors and recipients. The correlation between the two methods was y = 1.48 micrograms/L + 0.8x (r = 0.89). The bias (mean difference) was 12 micrograms/L (0.055 mumol/L) through the MEGX concentration range measured (0-250 micrograms/L, 0-1.136 mumol/L). We observed a major difference between the two methods in samples from four recipients and one donor. Cross-reactivity in FPIA with lignocaine and two of its metabolites (glycinexylidide and 2,6-xylidine) was < 3%. Samples with high bilirubin concentrations (> 200 mumol/L) required dilution before assay of MEGX by FPIA. Although there was an increase in apparent MEGX concentrations in some samples with increased bilirubin concentrations, the relationship was not constant. Increased plasma concentrations of cholesterol and triglyceride resulted in relatively small increases in apparent MEGX concentrations.


Lactate measured in diluted and undiluted whole blood and plasma: comparison of methods and effect of hematocrit. We evaluated a new analyzer that measures lactate in undiluted whole blood by direct (or undiluted) amperometry [Nova Stat Profile 7 Analyzer (SP7); Nova Biomedical, Waltham, MA] by comparing it with two other analyzers, one for measuring lactate in whole blood by indirect (or diluted) amperometry [Model 2300; Yellow Springs Instrument Co. (YSI), Yellow Springs, OH] and another for measuring lactate in plasma by enzymatic colorimetry (aca; Du Pont Co., Wilmington, DE). All between-method comparisons of the three methods showed that the results for plasma were comparable (Sy/x = 0.24-0.33 mmol/L). Within-method comparisons by the YSI differed substantially between plasma and whole blood (Sy/x = 0.48 mmol/L), but within-method comparisons by the SP7 produced better agreement between plasma and whole blood (Sy/x = 0.18 mmol/L). The difference between whole blood and plasma by YSI is related to hematocrit, with the greatest differences noted for samples with the highest hematocrit. Serum lactate measured by SP7 had between-day imprecision (CV) ranging from 12% at 0.5 mmol/L to 4.2% at 3.7 mmol/L, showed a linear standard curve to at least 11.5 mmol/L, and was independent of hematocrit. There was a mean bias of approximately 0.4 mmol/L for results in the reference range for both plasma and whole blood by SP7 compared with plasma results by either aca or YSI.


Direct determination of zinc in serum by Zeeman atomic absorption spectrometry with a graphite furnace. We developed a precise and accurate graphite furnace atomic absorption spectrometric method for the direct determination of zinc in serum. Serum samples are analyzed after 20-fold dilution with water of ultrapure analytical grade. No other reagent is used, from the moment of sampling until measurement. During atomization, the argon flow is kept at 150 mL/min instead of gas stop, to decrease the sensitivity and thus allow lower dilution ratios. Zinc concentrations are determined against a serum-matched calibration curve. Graphite tubes are uncoated and no L'vov platform is used. Between-run CVs were 5.9%, 3.5%, and 1.9% for serum zinc concentrations of 0.93, 1.15, and 1.43 mg/L, respectively. The characteristic mass was 9 pg, and the detection limit (meanblank + 3SDblank) was 0.060 mg/L.


Relation between magnesium and potassium concentrations in myocardium, skeletal muscle, and mononuclear blood cells. Potassium and magnesium were measured in 26 cardiac surgery patients (right atrial appendage), 23 autopsy subjects (right atrial appendage, left ventricular free wall, and skeletal muscle), and 9 healthy volunteers (mononuclear blood cells) to determine whether there was a relation between these two ions in the tissues measured. In the cardiac surgery patients, the potassium and magnesium concentrations were 46.35 +/- 3.89 and 4.40 +/- 0.58 (mean +/- SD, mumol/g wet weight tissue), respectively, and were significantly correlated (r = 0.54, P = 0.005). In the autopsy group, the respective concentrations were: for right atrial appendage, 30.54 +/- 10.18 and 3.66 +/- 0.70 mumol/g (r = 0.38, P = 0.14); left ventricular free wall, 60.69 +/- 17.93 and 7.74 +/- 1.73 mumol/g (r = 0.92, P = 0.0001); and skeletal muscle, 93.05 +/- 20.49 and 8.64 +/- 2.06 mumol/g (r = 0.91, P = 0.0001). In the healthy volunteer group, the results for potassium and magnesium in mononuclear blood cells were 42 +/- 9.9 and 3.99 +/- 0.70 fmol/cell, respectively (r = 0.94, P = 0.0001). Thus, potassium and magnesium concentrations were significantly correlated in all the tissues measured.


Studies of lipid peroxidation products in cerebrospinal fluid and serum in multiple sclerosis and other conditions. Lipid peroxides were measured in cerebrospinal fluid (CSF) and sera from patients with multiple sclerosis (MS) or other conditions and from control subjects. In serum, visible fluorescence, A240nm, and free and protein-bound thiobarbituric acid (TBA)-reactive substances were significantly greater in MS patients than in control subjects. No such differences were observed in CSF. There was no correlation between lipid peroxides and disease severity or relapse time. We observed a significant positive correlation between CSF protein-bound TBA-reactive substances and CSF protein, suggesting that production of the former depends on protein concentration. A significant negative correlation between free TBA-reactive substances in serum and ultraviolet fluorescence indicated that production of free TBA-reactive substances may be associated with decreased production of fluorescent lipid peroxides. This study provides evidence for increased lipid peroxidation in serum but not CSF from MS patients, suggesting that either there is no increase in CSF lipid peroxidation in MS patients or that CSF lipid peroxides are rapidly removed, possibly by binding to CSF proteins that can be reabsorbed into the blood.


Reference values for immunoglobulin kappa and lambda light chains and the kappa/lambda ratio in children's serum. We analyzed 708 serum samples from healthy children and adolescents by immunonephelometry to obtain reference values for the immunoglobulin kappa (kappa) and lambda (lambda) light chains and for their ratio at a time of life when immunoglobulin synthesis is maturing and continually being stimulated. The lambda chain concentration that is to be maintained throughout the child's life is reached very early, just after 1 year, whereas the concentration of the kappa chains, which increases gradually, reflects the concentration of the immunoglobulins as a whole. These reference values may be useful for studying kappa and lambda chains in illnesses involving the immune system in children.


Determination of beta 2-microglobulin in serum by a microparticle-enhanced nephelometric immunoassay. This fully automated nephelometric immunoassay to quantify beta 2-microglobulin in human serum measures the light-scattering signal produced by agglutination of commercially available latex microparticles (diameter 0.1 micron) coated with specific F(ab')2 against beta 2-microglobulin. The calibration curve, generated by serial dilutions of a beta 2-microglobulin standard of known concentration, is used to calculate beta 2-microglobulin concentrations in serum samples by the logit-log function and linear-regression analysis. The assay range (sample dilution 400-fold) extends from 0.3 to 40.0 mg/L. No antigen excess appears at beta 2-microglobulin concentrations up to 320 mg/L. Within-run CVs ranged from 1.0% to 3.4%, and between-days from 1.2% to 2.8%. Total imprecision (CV) was < 5%. Analytical recovery averaged 99.5% +/- 2.8%. Rheumatoid factor, complement, bilirubin (up to 340 mumol/L), and hemoglobin (up to 2.0 g/L) do not interfere. Strongly turbid lipemic samples must be cleared before analysis. Standard curve linearity was very good in samples covering the clinical useful range of concentrations. Results of the method correlated well with those of radioimmunoassay and microparticle enzyme-linked immunoassay (r = 0.979 and 0.975, respectively). The reference interval (nonparametric estimation) in apparently healthy adults (n = 303) was 0.87 (0.80-0.94) to 2.42 (2.28-2.45) mg/L; the median value was 1.54 mg/L.


Measurement of free desipramine in serum by ultrafiltration with immunoassay. We developed an ultrafiltration method for assaying free desipramine (DMI) in serum. An ultrafiltrate of DMI-containing serum was prepared by centrifugation through an Amicon Centrifree micropartition filter. Syva DMI solid-phase extraction (SPE) columns were used to extract the DMI from the serum and ultrafiltrate. The Syva monoclonal EMIT assay was used to quantify the DMI in the extract. In some experiments, the percent free DMI was quantified with radioactivity. Nonspecific losses of DMI in serum to the ultrafilter system were low (recoveries > 91%). Extraction of [3H]DMI from phosphate-buffered saline (to mimic serum ultrafiltrate) with the Syva SPE system was quantitative (recoveries of 98.4% +/- 4.6%). Free DMI concentrations, derived from serum containing 2.5-2500 micrograms/L DMI, were determined by ultrafiltration; results agreed well with values determined by equilibrium dialysis, the average percent of free DMI being 18.4% +/- 0.25% and 15.9% +/- 0.51%, respectively. To increase the sensitivity of the free DMI assay in the therapeutic range (total DMI 125-300 micrograms/L), we increased fourfold the ultrafiltrate volume applied to the SPE column. For free DMI at 11-130 micrograms/L, the within-run and between-run CVs for the ultrafiltration method were < 9% and < 15%, respectively. Binding of DMI to serum proteins decreased over the pH range 6.0-8.0, although temperatures between 20 and 28 degrees C did not affect binding. The ultrafiltration assay is fast, accurate, simple, and adaptable to standard laboratory instrumentation.


Interlaboratory standardization of measurements of glycohemoglobins. The diversity of methods used to measure glycohemoglobins (GHb) makes it difficult to compare patients' results among laboratories. We reported previously the feasibility of providing comparable results from different assays by use of common calibrators. We here compare results from seven different GHb methods calibrated by use of hemolysates assayed by a precise ion-exchange high-performance liquid-chromatographic (HPLC) method for hemoglobin A1c (HbA1c). Thus, regardless of the GHb species measured by the seven methods, results were referenced to the HbA1c content of the calibrators. Without this calibration, GHb values for single samples varied, e.g., from 4.0% to 8.1% and from 10% to 14.2% in the normal and high ranges, respectively. Calibration decreased between-method variability (single sample ranges of, e.g., 4.8% to 5.4% and 9.4% to 10.2% in the normal and high ranges, respectively) and improved interassay precision. We conclude that this approach to calibration of GHb measurements allows direct comparison of results obtained by different methods and improves precision.


Arsenic analysis II: rapid separation and quantification of inorganic arsenic plus metabolites and arsenobetaine from urine. We describe the rapid separation of inorganic arsenic plus metabolites from arsenobetaine or seafood arsenic in urine. Traditional, high-pressure liquid chromatography is replaced by disposable silica-based cation-exchange cartridges for this separation. Both fractions are quickly separated and collected for analysis by atomic absorption spectrophotometry. Analytical recovery of both fractions is > or = 95%, with an overall precision (CV) ranging from 1.6% to 6.4%. Using this method, we correctly identified the sources of arsenic exposure, whether of inorganic or seafood origin, in 11 urine specimens supplied by the Centre de Toxicologie du Quebec.


Perspectives in alkaline phosphatase research. Gene cloning and site-directed mutagenesis have had a profound effect on alkaline phosphatase research. Four distinct structural genes encoding placental, intestinal, and tissue-nonspecific isoenzymes have been cloned, sequenced, and mapped to human chromosomes. Differences in properties between the respective gene products are due to variations in primary structure involving only one, or a few, key amino acid residues. Recognition that alkaline phosphatase belongs to the category of molecules that are localized to cell membranes through a COOH-terminal glycan-phosphatidylinositol anchor provides a basis for understanding the generation of isoforms observed in plasma in disease. Isoforms produced by differential cleavage or preservation of the glycan-phosphatidylinositol anchor may offer new correlations with disease that are of diagnostic value. However, a more important contribution of alkaline phosphatase research to clinical chemistry may prove to be an increased understanding of disease processes at the molecular level.


The two mRNAs encoding rat intestinal alkaline phosphatase represent two distinct nucleotide sequences. Rat duodenal mucosa contains two mRNAs (2.7 and 3.0 kb) encoding intestinal alkaline phosphatase (IAP), but the mechanism for their production has not been clear. By means of the polymerase chain reaction (PCR), we isolated a fragment that identifies a second rat IAP (rIAP-II), differing from the rIAP-I sequence in the coding and 3' untranslated regions and encoding a COOH-terminal sequence predicted to be hydrophilic. By means of probes unique to each sequence, rIAP-I identified the 2.7-kb mRNA, and rIAP-II the 3.0-kb mRNA. The different structures and differential regulation of the mRNAs after fat feeding demonstrate the presence of two rat IAP transcripts.


Placental alkaline phosphatase: a model for studying COOH-terminal processing of phosphatidylinositol-glycan-anchored membrane proteins. Placental alkaline phosphatase (PLAP) has been used as a model for studying the biosynthesis of the phosphatidylinositol-glycan (PI-G)-protein linkage in intact cells and in cell-free systems. However, for the study of processing in cell-free systems, a small protein devoid of glycosylation sites is preferable. A PLAP-derived cDNA was engineered that codes for a nascent protein (mini-PLAP) of 28 kDa in which the NH2- and COOH-termini are retained but most of the interior of PLAP is deleted. In vitro translation of mini-PLAP mRNA in the presence of rough microsomal membranes yields mature PI-G-tailed mini-PLAP. Processing of nascent mutant proteins occurs only when a small amino acid is located at the site of cleavage and PI-G attachment (omega site). Mutations adjacent and COOH-terminal to the omega site have revealed that the omega + 1 site is promiscuous in its requirements but that only glycine and alanine are effective at the omega + 2 site. Rough microsomal membranes from T cells deficient in PI-G biosynthesis do not support processing of mini-PLAP; addition of exogenous PI-G restores activity. Translocation of the proprotein, most likely requiring ATP and GTP, precedes COOH-terminal processing.


Differential release of human intestinal alkaline phosphatase in duodenal fluid and serum. Human intestinal alkaline phosphatase (IAP) can be released by the enterocyte into duodenal fluid as a mixture of three isoforms. A proportion of the enzyme is associated with triple-layered membrane vesicles (vesicular IAP). Although, occasionally, free hydrophilic IAP dimers are present, the remaining enzyme usually consists of a mixture of hydrophobic IAP dimers and more complex hydrophobic IAP structures of larger size, both entities being identified as "intestinal variant" alkaline phosphatase (VAR IAP). The hydrophobicity of VAR IAP stems exclusively from its attached glycosyl-phosphatidylinositol (GPI) anchor. Both vesicular IAP and VAR IAP are converted to hydrophilic enzyme upon removal of the GPI tail by phospholipase D (PLD) present in duodenal fluid. The IAP released into the vascular bed consists mainly of VAR IAP; vesicular IAP is absent. The enzyme characteristics of VAR IAP partially purified from duodenal fluid and from serum are identical. In plasma, VAR IAP appears to associate with (lipo)protein complexes and is thus protected from further degradation by plasma PLD. Such complex formation may explain why, in the serum of a healthy reference population, VAR IAP was more abundant than hydrophilic dimeric IAP.


Chemical nature of intestinal-type alkaline phosphatase in human kidney. Approximately 10% of the alkaline phosphatase activity in human kidney is derived from the intestinal-type alkaline phosphatase isoform, which can be differentiated from adult intestinal alkaline phosphatase by selective reactivity with monoclonal antibodies. The NH2-terminal sequence of the renal intestinal-type alkaline phosphatase was shown to be identical to sequences of the adult and meconial alkaline phosphatases except for the NH2-terminal valine residue, which is missing in the renal intestinal-type enzyme. Incubation of purified meconial alkaline phosphatase with kidney homogenate resulted in removal of the NH2-terminal valine residue, indicating the presence of aminopeptidases in kidney that catalyze this hydrolysis. Furthermore, the oligosaccharide chains of the renal intestinal-type alkaline phosphatase were shown to differ from those of meconial and adult intestinal alkaline phosphatases, as revealed by lectin affinity chromatography. The heterogeneity of the intestinal-type alkaline phosphatase can therefore be generated both by partial peptide bond hydrolysis and differences in glycosylation.


Placental alkaline phosphatase as the placental IgG receptor. We review data from our studies of the physiological role of placental alkaline phosphatase (PLAP) and report that, in addition to functioning in catalysis, PLAP has the capacity to bind the Fc portion of human IgG. The dissociation constant for the interaction (3.86 mumol/L) indicates that the PLAP-IgG complex probably occurs in vivo. Furthermore, the electrophoretic and immunochemical properties of PLAP are identical to those of the purified placental Fc receptor. This receptor is believed to participate in the transfer of IgG molecules from the maternal circulation to the fetus during pregnancy. Studies with HEp2 cells show that PLAP is necessary for the internalization of IgG molecules. PLAP behaves, at least in this cell line, as an Fc receptor. The presence of large amounts of PLAP in clathrin-coated vesicles prepared from placenta strongly indicates that PLAP is involved in the endocytic machinery in this organ. We conclude that these results, taken together, suggest a novel biological role for PLAP.


Alkaline phosphatase isoenzyme patterns in malignant disease. Early treatment of patients with malignant disease and liver or bone metastasis may increase their survival time. We have used the activity patterns of liver and bone isoenzymes of alkaline phosphatase (ALP), separated by agarose gel electrophoresis, to detect early metastasis. We studied ALP isoenzyme patterns in a background population of 101 patients with no evidence of any disease that might influence this pattern; a healthy reference population (n = 330); and the following three groups of patients: 143 with malignant disease, 47 with nonmalignant liver disease, and 22 with nonmalignant bone disease. Cutoff and predictive values of liver ALP, high-molecular-mass (high-M(r)) ALP, and bone ALP were established for detecting liver and bone metastasis. The positive predictive value of liver and high-M(r) ALP was higher than that of total ALP in detecting liver metastasis, but liver and high-M(r) ALP did not enable us to differentiate between malignant and nonmalignant liver disease. Total ALP activity was of slightly more value than liver and high-M(r) ALP in enabling us to rule out liver metastasis. From bone ALP activity we could not distinguish between nonmalignant bone disease and bone metastasis. The negative predictive value of bone ALP in the diagnosis of bone metastasis was low, but its positive predictive value was high and superior to that of total ALP.


Bone metal content in patients with chronic renal failure. Bone accumulation of metals, other than aluminum (Al), in patients with chronic renal failure (CRF) has rarely been studied. We report the bone and blood levels of iron (Fe), vanadium (V), lead (Pb), Al and calcium (Ca) in 18 CRF patients on long-term hemodialysis and in 14 controls (C). Significant increments in mean blood Al (1,300%), V (160%) and Fe (60%) and decrease in blood Pb (-50%) were found in the patients with CRF. Renal subjects had higher bone concentrations (mean +/- S.E.M.; microgram/g of bone) of Al (CRF = 129.1 +/- 16.9; C = 12.6 +/- 2.9; P < 0.0001), Fe (CRF = 940.4 +/- 133.4; C = 263.4 +/- 49.0; P < 0.0001) and V (CRF = 2.55 +/- 0.43; C = 1.39 +/- 0.36; P = 0.0308) and lower Ca (CRF = 268.2 +/- 11.1 mg/g; C = 377.2 +/- 28.1; P = 0.0017). A positive correlation was found between bone Al and V (r = 0.355, P < 0.05). Results indicated that a significant bone accumulation of Al, Fe and V, but not Pb, occurred in the hemodialyzed azotemic individuals.


High-performance liquid chromatographic assay for L-glyceric acid in body fluids. Application in primary hyperoxaluria type 2. We describe a liquid chromatographic technique to determine L-glycerate in body fluids. The method is based on the derivatisation of the L-glycerate by incubation with lactate dehydrogenase and nicotinamide-adenine dinucleotide in the presence of phenylhydrazine. Oxidation of L-glycerate forms beta-hydroxypyruvate which is converted in turn into the related phenylhydrazone. The UV-absorbing derivative is determined using reversed-phase high performance liquid chromatography. The sensitivity was 5 mumol/l and 50 microliters of sample were required. The imprecision relative standard deviation was 4.5% and the recovery was 96.5 +/- 6.8% for L-glycerate in plasma. L-Glycerate concentrations in urine and plasma were less than 5 mumol/l in both normal individuals and patients with glycolic aciduria. In a patient with systemic oxalosis and normal plasma glycolate, plasma L-glyceric acid was 887 mumol/l.


Red blood cells and platelet membrane fatty acids in non-dialyzed and dialyzed uremics. Three groups of patients with chronic renal failure (CRF), 16 non-dialyzed, 16 undergoing haemodialysis (HD), 16 undergoing continuous ambulatory peritoneal dialysis (CAPD), and 48 controls were examined. We analyzed the fatty acid composition in membranes from erythrocytes and platelets and the platelet malondialdehyde (MDA) production as an index of thromboxane metabolism. Marked differences in erythrocytes fatty acid composition were observed between patients with CRF and controls and, particularly, among the three groups of patients with CRF. Patients on CAPD were characterized by an increase in oleic acid, while haemodialyzed had a marked increase in arachidonic acid. Platelet fatty acid composition showed similar differences, suggesting a 'systemic' membrane abnormality. Platelet MDA was increased in haemodialyzed and positively correlated with the platelet arachidonate content.


Distribution of lipoprotein species (LpA-I, LpA-I:A-II) in serum and HDL subfractions of untrained and trained normolipemic men. The distribution of lipoprotein species (LpA-I, LpA-I:A-II) in serum and within HDL subfractions (HDL2b, HDL2a, HDL3) was examined in 26 sedentary and 19 endurance trained normolipemic male individuals. The concentrations of lipids and apolipoproteins in serum and HDL subfractions and the concentrations of LpA-I and LpA-I:A-II were determined. Significant differences (Mann-Whitney-U-test) were found in serum concentrations of apoB (P < 0.05), apoA-II (P < 0.01) and LpA-I:A-II (P < 0.001). In HDL3 apoA-II concentration was significantly lower in the trained group (P < 0.05) but in HDL2 subclasses the concentrations of apoA-I and apoA-II did not differ between the groups. Despite similar concentrations of the two apolipoproteins, there were difference in the distribution of lipoprotein species within HDL2 subfractions. The concentrations of LpA-I did not differ, but the concentrations of LpA-I:A-II particles were higher in the trained group. Untrained and trained had similar concentrations of apoA-II (in HDL2b) but obviously more apoA-II containing particles and this leads to the assumption that within HDL2 of endurance trained individuals LpA-I:A-II particles have a lower apoA-II content compared with particles of untrained individuals. The data emphasize, that normolipemic individuals of different maximum oxygen uptake have a different distribution and composition of lipoprotein species (LpA-I, LpA-I:A-II).


Incidence of 'unsuspected' extranodal head and neck lymphoma. A series of 100 consecutive patients presenting with non-Hodgkin's lymphoma in the head and neck region was reviewed. An examination of the contribution made to the staging classification by an otolaryngological examination showed that one-third of patients presenting with cervical nodes had unsuspected extranodal disease in Waldeyer's ring. Overall 14% of patients undergoing an otolaryngological examination had their disease upstaged because of that examination.


Changes in immunologic response in tonsillectomized children. I. Immunosuppression in recurrent tonsillitis. The possible immunoregulatory role of the tonsils was studied by determining immunoglobulins IgG, A, M, E and factors C'3, C'4 and PFB of the complement system before and after tonsillectomy. The synthesis in vitro of IgG and IgM by lymphocytes stimulated with pokeweed mitogen was also measured. There were statistically significant differences between pre and post-operative levels of serum IgG, IgA and IgM, which decreased after surgery. Practically no change in the mean values of IgE and no significant differences in the levels of serum C'3, C'4, and PFB, were found. The in-vitro synthesis of both immunoglobulins (IgG, IgM) by lymphocytes increased significantly after tonsillectomy. Our results suggest that not only does tonsillectomy have no counterproductive effect on the immune system, but that, on the contrary, it seems to improve the immune response, since it appears to unblock the suppression to which the immune system was subject.


Changes in immunological response in tonsillectomized children. II. Decreased cellular response. The immunological cellular response was studied in 22 children aged between 5 and 10 years. One week before and one month after tonsillectomy, the mitogenic response to phytohaemagglutinin (PHA), concanavalin (ConA) and pokeweed mitogen (PWM) was tested. Our results showed statistically significant decreases in stimulation index (SI) to PHA, ConA and PWM after tonsillectomy. This suggests that the tonsils may influence the cellular immune status in children. The higher level of preoperative activity in children with recurrent tonsillitis could be understood as the result of stimulation of lymphocytes by bacteria.


The role of mucosal receptors in the nasal sensation of airflow. 50 subjects were admitted into a randomized double-blind placebo controlled cross-over trial with 4% lignocaine as the active drug and normal saline as the placebo. Each subject had 2 ml of solution sprayed into each nasal cavity and all subjects had both sprays but on different occasions. The order in which the sprays were administered was randomized. The subjective sensation of nasal airflow was measured using a visual analogue scale before and after the spray. These measurements were made under conditions of the same airflow rate, which was monitored throughout the experiment using a reprogrammed NR6 rhinomanometer. Objective nasal patency was measured as peak nasal inspiratory flow rate. It was found that the nasal sensation of airflow decreased slightly after both lignocaine (difference between medians 5.0; 95% confidence interval -2.91 to 6.11) and normal saline (difference between medians 6.0; 95% confidence interval -1.02 to 7.21). Nonparametric analysis of variance showed this difference to be non-significant (P = 0.73). In addition there was no significant change in objective nasal patency. The results suggest that nerve endings in the nasal mucosa play no part in sensing nasal airflow during respiration.


Nasal airflow receptors: the relative importance of temperature and tactile stimulation. The receptors responsible for the nasal sensation of airflow have not been identified with certainty. Although both mechanoreceptors and thermoreceptors have been implicated, evidence suggests that the nose is more sensitive to cold air than to air at body temperature. The present study was designed to investigate the relationship between the velocity and the temperature of an airjet as regards its ability to stimulate the nasal lining. Both the nasal vestibule and the nasal cavum are more sensitive to cold air than to air at mean intranasal temperature (P < 0.001). A similar effect is seen with warm air which is as stimulating as cold air. The nasal vestibule is twice as sensitive as the nasal cavum to an airjet at mean intranasal temperature (P < 0.001). It is concluded that the nasal vestible is very sensitive to the tactile stimulation of an airjet. This effect is highly temperature dependent being much more pronounced for air temperatures above or below the mean intranasal temperature. The temperature effect is relatively more important in the nasal cavum which is very much less sensitive to stimulation than the vestibule.


T3 laryngeal cancer: a retrospective study of the Dutch Head and Neck Oncology Cooperative Group: study design and general results. 511 Patients with T3 N0-3 M0 squamous cell carcinoma of the larynx, treated in the Netherlands from 1975 until 1984, were retrospectively analysed. Four different treatment policies were followed: primary surgery, planned combination of radiotherapy and surgery, primary radical radiotherapy, and selective radiotherapy. General results are presented. Local control rate was 72%. Regional control rate was 90% for clinically N0 patients and 78% for clinically N+ patients. Salvage therapy was overall successful in 38%. Surgical salvage for local radiation failures (with regional relapse) was successful in 69%, and for regional failures (without local relapse) in 46%. Ultimate locoregional control was 78% and, due to 8% distant metastases, 5-year actuarial corrected survival was 70%. Prognosis did not improve over the years. Corrected survival was independently correlated with tumour extension, involvement of neck nodes and treatment strategy. Corrected survival was similar for primary radiotherapy and primary surgery, but significantly better for planned combined therapy. Multiple primary tumours occurred significantly more often in male (19.5%) than in female patients (7.3%) (P = 0.05), the bronchus being most commonly affected. Cumulative actuarial risk for metachronous tumour was 15% after 5 years and 30% after 10 years so prevention and early detection of these second tumours may play the most important role in improving overall survival rates in the future.


The role of calcium alginate swabs in adenotonsillectomy. A randomized controlled study using swabs of calcium alginate versus gauze was undertaken to evaluate the haemostatic capabilities and the shortening of operative time. Haemostasis in adenoidectomy was shown to be more effective and the operating time shortened (P = 0.05) by the use of calcium alginate. Haemostasis and operating time for tonsillectomy were not affected.


Non-surgical management of acoustic neuromas. Watchful waiting is one of the options available in the management of acoustic neuromas and this article deals with 13 patients who were so managed. Non-operative management was advised because of age, poor general health, small size of tumour, only hearing ear, or in patients unwilling to undergo surgery for various reasons. This group was followed up at 6-12-monthly intervals and the follow-up period ranged from 1 to 18 years (mean 5.3 years). Ten patients had small tumours and only in 2 of these was increase in tumour size demonstrated on follow-up CT scan. In one this increase was later followed by regression. Two patients required partial removal of tumour because of increasing symptoms after 3 and 7 years of follow-up; one of them died on the twelfth post-operative day. There appears to be a small group of patients for whom delay is worth while rather than to subject all patients with acoustic neuroma to surgery from which full recovery cannot be guaranteed.


The effect of parental smoking on outcome after treatment for glue ear in children. A sample of 201 children aged between 2 and 9 years with bilateral chronic otitis media with effusion (OME) were treated prospectively and at random by adenoidectomy, adenotonsillectomy, or with neither procedure. In all cases only a unilateral grommet was inserted and the contralateral unoperated ear was examined one year post-operatively for persistence or resolution of the effusion. A self-administered questionnaire was completed by the parents concerning their smoking habits. The resolution of effusion following surgery was assessed in relation to smoking by by the mother and father separately and in combination. Clearance of glue was statistically less frequent where the child's mother or where both parents smoked. This was related to the number of cigarettes smoked by the mother or both parents. The adverse effect was demonstrable whether or not adenoidectomy or adenotonsillectomy had been performed for treatment. The findings lend further support or professional and governmental opinions of a deleterious effect of passive smoke exposure on children and in this case parental smoking has been shown to have an adverse effect on the outcome of OME following surgical treatment.


Results of full-thickness laryngotracheal wall reconstruction: a survey of the literature. Full-thickness reconstruction of the laryngotracheal wall is needed after tumour removal, and also in patients where lumen augmentation is required to resolve laryngotracheal stenosis. For repairing such defects, several techniques are available. All techniques are more successful when small defects are reconstructed and less successful when there is a major deficit. The main problem with most methods of reconstruction is their unreliable blood supply which becomes most obvious in repairing larger areas.


Computed tomography (CT) and magnetic resonance imaging (MRI) in T-stage evaluation of oral and oropharyngeal carcinomas. 174 CT and 32 MRI investigations were carried out on patients with carcinomas of the mouth and oropharynx. The methods were compared and their value in predicting the pre-therapeutic T-staging studied. The combination of CT and clinical examination was able to improve the T-staging considerably compared with clinical examination alone. MRI was superior to CT in delineating the tumour margins in 78% of patients. T1-weighted and gradient echo sequences after intravenous gadolinium injection were particularly useful. Nevertheless, T-staging based on clinical and CT-findings was changed in only 5% of patients by MRI. MRI is especially useful in T1 tumours and in cancer of the base of the tongue. MRI should also be used instead of CT were dental fillings obscure the region of interest. If there are good MRI facilities and an experienced team available MRI can be used before CT.


CO2-laser treatment of recurrent glottic carcinoma. The records of 23 patients treated by CO2-laser surgery for recurrent glottic carcinoma after radiotherapy were studied retrospectively to assess treatment results and complications. 15 patients (65%) were cured with one or more laser procedures. The remaining 8 underwent a total laryngectomy for a second or third recurrence. One patient (4%) died of disease. No major complications from CO2-laser surgery were encountered. It is concluded that CO2-laser surgery is a safe method of treatment of recurrent glottic carcinoma.


Treatment of T3 carcinoma of the larynx by surgery or radiotherapy. T3 carcinoma of the larynx may be treated by surgery or by irradiation. A large number of oncologists feel that radical surgery offers a better chance of cure than does radiotherapy. We report a series of 147 patients with T3 N0 carcinoma of the larynx treated either by irradiation with salvage laryngectomy in the event of a recurrence, or by total laryngectomy. 109 patients had radiotherapy and 38 patients underwent surgery. The groups were well matched with no significant difference between the significant prognostic factors. The 5-year survival rate between the radiotherapy (70%) and surgery (40%) groups was significantly different (chi 2 = 4.48, d.f. = 1, P < 0.05). In spite of the well known problems associated with retrospective studies the present series suggests that radiotherapy combined with salvage surgery is an oncologically sound option for treating T3 laryngeal carcinoma.


The use of ventilation tubes and the incidence of cholesteatoma surgery in the paediatric population of Liverpool. Many surgeons believe that grommet insertion has reduced the incidence of cholesteatoma and thus radical mastoidectomy. Several causes for this trend have been postulated, including the increased use of ventilation tubes to treat otitis media with effusion. We have studied the number of operations for cholesteatoma, together with the number of ventilation tube insertions in the paediatric population of Liverpool, over a 28-year period between 1963 and 1990. There was a decline in the number of operations for cholesteatoma and an increase in the use of ventilation tubes, but there was no significant correlation between the two. This suggests that other factors are responsible for the decline in surgery for cholesteatoma.


The quantitative analysis of dysphonia.

The quantitative analysis of dysphonia. A clinical method of measuring 'hoarseness' is presented. Its value is assessed in 3 different types of vocal cord lesion and compared against normal values. There are shown to be significant differences between the normal and the abnormal voice and also differences between each type of vocal cord lesion. We feel that this quantification of hoarseness is useful in the treatment of patients as well as in monitoring their progress under therapy.


Salvage laryngectomy after radical radiotherapy for laryngeal carcinoma. Of 376 patients who were treated by radical radiotherapy for squamous carcinoma of the larynx, 56 subsequently underwent total laryngectomy. Residual or recurrent tumour was identified in 43 of the resection specimens, and necrosis alone in 13 cases, although a positive biopsy had been obtained in 3 of these prior to salvage laryngectomy. No disease related factors such as site or stage of the original tumour, or treatment related factors such as radiation type or dose, were found to be predictive of whether or not tumour was present. The clinical opinion of an experienced surgeon was found to have a positive predictive value of 0.86 for the presence of tumour. The fistula rate of salvage laryngectomy, 15 out of 56, was similar to that of other series. The actuarial cause specific 5-year survival for patients with tumour was 0.589, and for patients with necrosis only was 0.923. Intercurrent, smoking related disease was the cause of death in 16 of the 33 patients who have died.


Fundamentals of skin cancer/melanoma screening campaigns. There is increased world-wide concern about the rising incidence of melanoma and non-melanoma skin cancer. Screening theoretically reduces death and morbidity from skin cancer/melanoma. Visual examination of the skin is a rapid, safe and inexpensive screening tool. In this review the fundamentals of early disease detection before implementation of a public-health screening programme are critically analysed with reference to the skin cancer/melanoma, epidemic. It is concluded that skin cancer/melanoma fulfils, for the most part, the criteria enunciated by Wilson and Jungner in 1968. However, information about the effect of screening on reducing incidence and mortality is still lacking. Future research should focus on methods of improving compliance and on the costs and benefits of such screening programmes.


The application of the seven-point check-list in the assessment of benign pigmented lesions. One hundred consecutive histologically benign lesions seen at a pigmented lesion clinic were studied prospectively to assess the diagnostic accuracy of the seven-point check-list. Lesions were scored by both patient and physician. Thirty per cent of lesions attained a suspicious score. While there was a high level of agreement between patient and clinician regarding the individual characteristics of a lesion as defined in the check-list, final assessments differed. Most patients considered their lesions suspicious, whereas the clinicians correctly recognized that in 94% cases they were benign. The seven-point check-list often fails to exclude common benign pigmented lesions. The revised (1989) check-list is even less specific (70% of lesions achieving a suspicious score). Such check-lists are difficult for the inexperienced to interpret. Clinical familiarity is required in order to provide a confident diagnosis. Future emphasis should be directed towards improving screening in primary care.


Cutaneous metastases from breast carcinoma: a report of 18 cases. In this study, the authors report 18 patients with metastatic breast carcinoma affecting the skin, observed since 1988 in Forli, Italy. The results are compared with those already published in the literature.


Skin thickness in psoriasis.

Skin thickness in psoriasis. Skin thickness measured by the Harpenden skin-fold caliper showed that the uninvolved or non-lesional skin thickness in psoriasis does not differ significantly from that of normal skin. It is thinner in females than in males under 65 years and decreases with age in both sexes. It is thinner in those with fair skin compared to those who tan easily. Uninvolved skin thickness in psoriasis appears to influence the thickness of an adjacent psoriatic plaque.


Health-care professionals' views of the effectiveness of pressure ulcer treatments. A survey among nursing-home physicians, dermatologists and nursing staff in The Netherlands. In a survey, 237 nursing-home physicians, 113 dermatologists, and 164 supervisory nurses in nursing homes rated 30 treatments for stage III pressure ulcers by scoring their effectiveness. On average, there seems to be a fair degree of consensus between these groups of health-care workers about the effectiveness of most treatments. The most notable exceptions are ultraviolet light, ultrasound, and laser therapy and myocutaneous flap operations. Here, the dermatologists disagree with both nursing-home physicians and nurses. However, within all specialties there is disagreement about the efficacy of ultraviolet light, ultrasound, and laser therapy and the topical application of zinc oxide, betadine iodine, sodium hypochlorite and a few others.


Localized crusted scabies in the acquired immunodeficiency syndrome. Crusted scabies (CrS) is an uncommon occurrence among patients with AIDS. Indeed to date only five cases have been described, all with widespread lesions. A case of localized CrS appearing as a yellowish and crusted plaque on the second right toe is reported in a woman with AIDS. Scraping off the verrucous surface, as well as punch-biopsy revealed many mites within the horny layer. The infestation is related to the cutaneous immune response and thus CrS should be considered an opportunistic infestation in AIDS. The importance of the early diagnosis of CrS in order to prevent disseminated lesions and involvement of other health-care workers is underlined.


The use of calcipotriol in HIV-related psoriasis. We report the case of a 28-year-old homosexual man with advanced HIV disease (CDC classification group IV A) who developed erythrodermic psoriasis which responded to calcipotriol, a topical vitamin D analogue. We believe this to be the first reported case of HIV-related psoriasis responsive to this form of treatment. Systemic therapy for HIV-related psoriasis is limited because of immunosuppressive effects. We suggest that calcipotriol may prove to be a useful therapeutic option in these patients.


Familial tufted angioma.

Familial tufted angioma. Tufted angioma is a rare slowly progressive vascular lesion found typically in children and young adults. Cases appear sporadically and clinically have been confused with low grade angiosarcoma, Kaposi's sarcoma, and multiple glomus tumours. We report a case of a patient with tufted angioma in whom a strong family history of similar lesions is apparent, transmitted in an autosomal-dominant mode of inheritance.


Lichen planus follicularis tumidus with cysts and comedones. The lichen planus follicularis tumidus was described by Belaich et al. in 1977, and we have found 13 cases published so far. We report two cases we have recently seen, one of them with multiple lesions on the head, and the other with a temporo-frontal lesion.


Localized familial redundant scalp: atypical cutis verticis gyrata? Two patients, a mother and son, with a conspicuous redundant unique scalp fold are reported. No underlying disorder was found. Light microscopy was unremarkable. An atypical familial form of cutis verticis gyrata is suggested.


Familial acne fulminans.

Familial acne fulminans. We report a patient of acne fulminans with arthralgia whose sister presented with the same disease at an identical age 10 years earlier. The two affected siblings in our family also had identical HLA phenotypes which further supported the role of genetic susceptibility in acne fulminans.


Bullous and haemorrhagic lichen sclerosus with scalp involvement. We describe a patient who developed a generalized blistering eruption due to lichen sclerosus and who was observed to have scalp involvement. Both are unusual manifestations of this disease which merit consideration. Lichen sclerosus is an uncommon disease that most frequently affects the external genitalia of perimenopausal women. The aetiology is unknown. Approximately 20% of affected patients have extragenital lesions that present as small, ivory, shiny round macules or papules that later become atrophic; extragenital lesions are generally asymptomatic. Bullous and haemorrhagic forms may occur but these are generally localized and reports of extensive or generalized involvement are rare. We describe an elderly woman with generalized bullous lichen sclerosus. As an incidental finding, she was observed to have lichen sclerosus affecting her scalp. This has rarely been described and it would appear that she is the third reported case of scalp involvement.


Possible induction of malignant melanoma by sunbed use. Malignant melanoma of the female genitalia is rare. A patient who regularly used a sunbed developed a malignant melanoma after 4 years. The possible aetiological role of sunbeds in the development of such a malignancy is discussed.


Perianal basal cell carcinoma.

Perianal basal cell carcinoma. Perianal basal cell carcinoma (BCC) is a rare tumour and only a few case reports can be found in the literature. In this location, it is important to differentiate a BCC from a basaloid cloacogenic carcinoma, as the first is an invasive but localized tumour, while the second has a high capacity for metastasis.


Epidermolysis bullosa acquisita presenting with localized facial blisters. A 44-year-old woman presented with blisters on her face of 1-month's duration. A biopsy showed a subepidermal bulla with a dermal infiltrate of inflammatory cells and thick linear deposits of immunoreactants along the dermo-epidermal junction. Using Western immunoblots, the patient's IgG antibody was found to recognize type VII procollagen. Moderate doses of oral prednisolone resulted in a complete remission, without a recurrence for more than 3 years.


Malignant melanoma complicated by schwannoma. We report two cases of malignant melanoma associated with a schwannoma. Case 1 is a 32-year-old Japanese man who had a nodular melanoma in his epigastrium, associated with one subcutaneous nodule on the neck and another on the right thigh, both of which were histologically-proven schwannomas. Case 2 is a 48-year-old Japanese woman with an acral lentiginous melanoma on her right sole who later developed a schwannoma of the cerebello-pontine angle. Of all 146 malignant melanoma cases found in our clinic during the past 20 years, 1.4% were associated with a neurogenic tumour, a significant association.


The development of scar sarcoidosis at the site of desensitization injections. Tender nodules may develop at the site of injection of allergen extracts. Some cases can be related to the aluminium contained in the preparation. We report a case where scar sarcoidosis developed at the site of desensitization injections. At presentation, there were no other cutaneous signs of sarcoidosis; systemic signs only developed 2 years later. We suggest that sarcoidosis be considered as a cause of granulomata developing at sites of desensitizing injections, particularly when aluminium is not involved.


Hypopigmented mycosis fungoides.

Hypopigmented mycosis fungoides. We report the case of a 25-year-old Jamaican woman with hypopigmented mycosis fungoides. She first developed a hypopigmented patch on her arm at the age of 11 years. Further lesions developed on the trunk and limbs over a period of 10 years. The lesions were completely impalpable. Skin biopsy showed an infiltrate of atypical lymphocytes, some with cerebriform nuclei, suggesting a diagnosis of mycosis fungoides. The lesions cleared with PUVA therapy.


Acrodermatitis continua responding to cyclosporin therapy. A 69-year-old man with painful, recalcitrant, localized acrodermatitis continua developed widespread pustulation which was resistant to high-dose methotrexate therapy. Low-dose cyclosporin induced a rapid and persistent remission.


Secondary T-cell lymphoma presenting as a giant ulcer. Secondary cutaneous T-cell lymphoma may present with various types of skin lesions, but rarely produces ulceration in contrast to primary cutaneous T-cell lymphoma. We report a case of malignant lymphoma of the tonsil that recurred as a giant cutaneous ulcer on the back of a 45-year-old man. Biopsy of the ulcer revealed a malignant lymphoma of diffuse, mixed-cell type. Surface marker analysis of the tumour cells showed suppressor/cytotoxic T-cell characteristics. The skin lesions were treated by electron beam therapy.


Morphological changes associated with growth inhibition of Trichophyton mentagrophytes by amorolfine. Amorolfine inhibited the in-vitro growth of Trichophyton mentagrophytes to some extent at a low drug concentration of 0.8 ng/ml. Corresponding to the growth inhibition, SEM studies revealed a slight modification of hyphal morphology, i.e. a waving of the hyphal surface. These morphological alterations were more extensive with increases in drug concentration and treatment period: collapsed and distorted hyphae and exfoliation of the surface of T. mentagrophytes occurred at 8 ng/ml and marked deformation and disruption of the hyphal structure at 80 ng/ml of amorolfine. TEM revealed thickening of the cell walls and the accumulation of electron-dense granular structures in both the wall and cytoplasm in thin-sectioned cells pretreated with 8 ng/ml or more of amorolfine, although the nuclear and mitochondrial architecture was not noticeably influenced. Cytoplasmic membranes and other membranous structures of organelles such as nuclei and mitochondria were disrupted or fused, thereby losing their essential physiological activity in hyphal cells pretreated with 80 ng/ml of amorolfine. The ultrastructural study thus supports the observation that morphological changes of T. mentagrophytes caused by amorolfine were associated with its growth-inhibitory and killing activity, which depended on the drug concentration and treatment time.