The role of the clinical biochemist in detection of zinc-induced copper deficiency.

A middle-aged woman with neutropenia and ataxia was found to have raised plasma zinc and profoundly low plasma copper concentrations. When found that she had been prescribed 135 mg zinc/day for seven years, a diagnosis of zinc-induced copper deficiency was made. After the zinc prescription was stopped, her copper and zinc concentrations and neutropenia normalized but she only had partial improvement in neurological status. The diagnosis of zinc-induced copper deficiency can be facilitated by the laboratory through measurement of plasma zinc concentration in patients with a low plasma copper concentration.

The kinetic basis of a general method for the investigation of active site content of enzymes and catalytic antibodies: first-order behaviour under single-turnover and cycling conditions.

The theoretical foundation has been laid for the investigation of catalytic systems using first-order kinetics and for a general kinetic method of investigation of the active site content, E(a), of enzymes, catalytic antibodies, and other enzyme-like catalysts. The method involves a combination of steady-state and single-turnover kinetics to provide Vmax and Km and k(lim)(obs) and K(app)(m), respectively. The validity of the method is shown to remain valid for two extensions of the simple two-step enzyme catalysis model (a) when the catalyst preparation contains molecules (Eb) that bind substrate but fail to catalyse product formation and (b) when the catalyst itself binds substrate non-productively as well as productively. The former is a particularly serious complication for polyclonal catalytic antibodies and the latter a potential complication for all catalysts. For the simple model and for (b) Vmax/k(lim)(obs) provides the value of [Ea]T and for (a) its upper limit. This can be refined by consideration of the relative values of Km and the equilibrium dissociation constant of EbS. For the polyclonal catalytic antibody preparation investigated, the fact that K(app/m) > Km demonstrates for the first time the presence of a substrate-binding but non-catalytic component in a polyclonal preparation. First-order behaviour in catalytic systems occurs not only with a large excess of catalyst over substrate but also with lower catalyst/substrate ratios, including the equimolar condition, when K(app)(m) >> [S]0, a phenomenon that is not widely appreciated.

Choline acetyltransferase, acetylcholinesterase, and nicotinic acetylcholine receptors of human gingival and esophageal epithelia.

A non-neuronal cholinergic system that includes neuronal-like nicotinic acetylcholine receptors (nAChRs) has recently been described in epithelial cells that line the skin and the upper respiratory tract. Since the use of nicotine-containing products is associated with morbidity in the upper digestive tract, and since nicotine may alter cellular functions directly via nAChRs, we sought to identify and characterize a non-neuronal cholinergic system in the gingival and esophageal epithelia. mRNA transcripts for alpha3, alpha5, alpha7, and beta2 nAChR subunits, choline acetyltransferase, and the asymmetric and globular forms of acetylcholinesterase were amplified from gingival keratinocytes (KC) by means of polymerase chain-reactions. These proteins were visualized in the gingival and esophageal epithelia by means of specific antibodies. Variations in distribution and intensity of immunostaining were found, indicating that the repertoire of cholinergic enzymes and receptors expressed by the cells changes during epithelial maturation, and that an upward concentration gradient of free acetylcholine exists. Blocking of the nAChRs with mecamylamine resulted in reversible loss of cell-to-cell adhesion, and shrinking and rounding of cultured gingival KC. Activation of the receptors with acetylcholine or carbachol caused stretching and peripheral ruffling of the cytoplasmic aprons, and formation of new intercellular contacts. These results demonstrate that both the keratinizing epithelium of attached gingiva and the non-keratinizing epithelium lining the upper two-thirds of the esophageal mucosa possess a non-neuronal cholinergic system. The nAChRs expressed by these epithelia are coupled to regulation of cell adhesion and motility, and may provide a target for the deleterious effects of nicotine.

Suppression of experimental autoimmune tubulointerstitial nephritis in BALB/c mice by berberine.

Berberine (BB) is a protoberberine alkaloid derived from various representatives of the Berberidaceae family. Although used as a therapeutic agent, it has not been applied in the treatment of immune-mediated disorders. In the present study, BB was administered at a daily dose of 10 mg/kg for 3 consecutive days before the induction of tubulointerstitial nephritis (TIN) by injection of bovine tubular basement membrane (TBM) antigen in BALB/c mice. The animals were investigated 2 months after TBM inoculation. The intensity of pathological injuries in animals with TIN+BB decreased significantly, an effect that correlated with the improvement of renal function. Flow cytometric analysis of peripheral blood cells showed that BB caused a decrease in the number of CD3(+), CD4(+), CD8(+), and sIg(+) lymphocytes in comparison with TIN mice. The same tendency was noticed in the lymphocytes from kidney infiltrates of treated animals. The control animals treated only with BB showed a decrease in the number of CD3(+), CD4(+), CD8(+) T-lymphocytes in comparison with control nontreated mice. Our results, thus, indicate that BB has an immunosuppressive effect in the TIN model, which is an analogue of various human kidney autoimmune diseases.

A general kinetic approach to investigation of active-site availability in macromolecular catalysts.

A potentially general kinetic method for the investigation of active-site availability in preparations of macromolecular catalysts was developed. Three kinetic models were considered: (a) the conventional two-step model of enzyme catalysis, where the preparation contains only active catalyst (E(a)) and inert (i.e. non-binding, non-catalytic) material (E(i)); (b) an extension of the conventional model (a) involving only E(a) and E(i), but with non-productive binding to E(a) (in addition to productive binding); (c) a model in which the preparation contains also binding but non-catalytic material (E(b)), predicted to be present in polyclonal catalytic antibody preparations. The method involves comparing the parameters V(max) and K(m) obtained under catalytic conditions where substrate concentrations greatly exceed catalyst concentration with those (klim/obs, the limiting value of the first-order rate constant, k(obs), at saturating concentrations of catalyst; and Kapp/m) for single-turnover kinetics, in which the reverse situation obtains. The active-site contents of systems that adhere to model (a) or extensions that also lack E(b), such as the non-productive binding model (b), may be calculated using [E(a)](T)=V(max)/klim/obs. This was validated by showing that, for alpha-chymotrypsin, identical values of [E(a)](T) were obtained by the kinetic method using Suc-Ala-Ala-Pro-Phe-4-nitroanilide as substrate and the well-known 'all-or-none' spectroscopic assay using N-trans-cinnamoylimidazole as titrant. For systems that contain E(b), such as polyclonal catalytic antibody preparations, V(max)/klim/obs is more complex, but provides an upper limit to [E(a)](T). Use of the kinetic method to investigate PCA 271-22, a polyclonal catalytic antibody preparation obtained from the antiserum of sheep 271 in week 22 of the immunization protocol, established that [E(a)](T) is less than approx. 8% of [IgG], and probably less than approx. 1% of [IgG].

Synthesis of a transition state analogue for the hydrolysis of cocaine: assistance to phosphonylation of a 3beta-hydroxytropane by a neighbouring amide group.

A simple synthesis of phenylphosphonate monoester analogues of the transition state for hydrolysis of the benzoyl ester group in cocaine is provided by the reaction of 2beta-amido-3beta-tropanols with phenylphosphonyl dichloride. Steric hindrance to phosphonylation of the hydroxyl is overcome because the neighbouring 2beta-amido group participates in the reaction. The intramolecular assistance by the amide to formation of the phosphonate ester is influenced by the electronic environment of the amide group.

Is atopic dermatitis a predisposing factor for experimental acute irritant contact dermatitis?

Proclivity to acute irritant contact dermatitis has been reviewed by comparing the response in patients with atopic dermatitis to controls. Although several controlled studies demonstrate such a proclivity, others do not, suggesting that the mechanisms involved are complex.

Characterization of the hydrolytic activity of a polyclonal catalytic antibody preparation by pH-dependence and chemical modification studies: evidence for the involvement of Tyr and Arg side chains as hydrogen-bond donors.

The hydrolyses of 4-nitrophenyl 4'-(3-aza-2-oxoheptyl)phenyl carbonate and of a new, more soluble, substrate, 4-nitrophenyl 4'-(3-aza-7-hydroxy-2-oxoheptyl)phenyl carbonate, each catalysed by a polyclonal antibody preparation elicited in a sheep by use of an analogous phosphate immunogen, were shown to adhere closely to the Michaelis-Menten equation, in accordance with the growing awareness that polyclonal catalytic antibodies may be much less heterogeneous than had been supposed. The particular value of studies on polyclonal catalytic antibodies is discussed briefly. Both the kcat and kcat/K(m) values were shown to increase with increase in pH across a pKa of approx. 9. Group-selective chemical modification studies established that the side chains of tyrosine and arginine residues are essential for catalytic activity, and provided no evidence for the involvement of side chains of lysine, histidine or cysteine residues. The combination of evidence from the kinetic and chemical modification studies and from studies on the pH-dependence of binding suggests that catalysis involves assistance to the reaction of the substrate with hydroxide ions by hydrogen-bond donation at the reaction centre by tyrosine and arginine side chains. This combination of hydrogen-bond donors appears to be a feature common to a number of other hydrolytic catalytic antibodies. High-pKa acidic side chains may be essential for the effectiveness of catalytic antibodies that utilize hydroxide ions.

Development of enzyme immunoassay for endogenous ouabain-like compound in human plasma.

Widespread evidence supports the existence of an endogenous digitalis-like compound in mammals. We report here the development of a novel enzyme immunoassay for ouabain that, in conjunction with a detailed HPLC study, identifies a ouabain-like compound (OLC) in extracted human plasma. The assay is sensitive-minimum detection limit for OLC 37 pmol/L (11 pmol/L in plasma)-and has a working range (between-assay CV < 10%) of 180-10000 pmol/L (54-3000 pmol/L in plasma). Mean recoveries of ouabain added to plasma ranged from 90% to 100%, and plasma extracts diluted in parallel to the standard curve. Plasma OLC concentrations in 10 healthy volunteers averaged 92 pmol/L (range 55-168), assuming 100% cross-reactivity of OLC in the ouabain assay. HPLC analysis with two distinct chromatographic conditions demonstrated that endogenous human plasma OLC co-eluted with authentic ouabain. The enzyme immunoassay is rapid and easy to perform and will support further investigation of the nature of this controversial endogenous steroid.

Detection of phencyclidine in urine using a polarization fluoroimmunoassay.

A polarization fluoroimmunoassay was developed for the detection of phencyclidine in urine and the reagents were adapted for use on the Abbott TDx analyser. The assay was used to look for evidence of phencyclidine abuse, over a 6 month period, amongst patients attending drug abuse clinics in the East End of London. Although over 2000 patients' samples tested negative, the assay successfully detected phencyclidine in an external quality control scheme.

A potential vaccine for cocaine abuse prophylaxis?

O. Bagasra, L.J. Forman, A. Howeedy, and P.A. Whittle recently conveyed the preparation of a cocaine immunogen that is a potential vaccine for cocaine abuse prophylaxis (Immunopharmacology: 1992, 23, 173-179). These investigators claim to have prepared a cocaine immunogen via reaction of cocaine with sodium metaperiodate followed by carrier protein, and to have generated anti-cocaine antibodies by using this immunogen. Periodate is used to conjugate haptens that contain a vicinal diol (or similar) group to carrier proteins and would not be expected to couple cocaine to protein. The authors of the above publication reported titres for antibodies generated by using their immunogen, but did not describe competitive inhibition of antibody binding by cocaine and did not report the specificity characteristics of their antibodies. The present author is not convinced by the claims made by Bagasra et al.

Catalytic antibody activity elicited by active immunisation. Evidence for natural variation involving preferential stabilization of the transition state.

1. The hydrolytic activity of IgG purified from (a) 13 samples of ovine antiserum collected from three animals during a two-year immunisation programme using a phosphate immunogen (comprising the amide conjugate bonded through the carboxy group of 4-nitrophenyl 4-carboxymethylphenyl hydrogen phosphate and amino groups of keyhole-limpet haemocyanin) and (b) a sample of ovine antiserum collected from another animal during an 18-week immunisation programme using an analogous sulphone immunogen (comprising the amide conjugate bonded through the amino group of 4-nitrobenzyl, 4-(4-aminobutoxy)benzyl sulphone and carboxyl groups of keyhole-limpet haemocyanin) were evaluated kinetically by using 4-nitrophenyl 4-(3-aza-2-oxoheptyl)phenyl carbonate and 4-nitrophenyl 4-(2-hydroxyethoxy)phenyl carbonate as substrates. 2. Catalytic activity was found in all 13 samples of anti-phosphate IgG but was absent in the sample of anti-sulphone IgG as well as in all samples of IgG isolated from the serum of non-immunised animals. These findings, taken together with the lack of catalytic activity of the anti-phosphate IgG towards the 2-nitrophenyl 4-(3-aza-2-oxoheptyl)phenyl carbonate, compel the view that the catalytic activity of the anti-phosphate IgG preparation is entirely antibody-mediated and is not due to contaminant hydrolytic enzymes. The fact that catalytic activity was found in all 13 samples of the anti-phosphate IgG provides the first evidence that it is possible, as a routine, to elicit a catalytic antibody response in a host animal via active immunisation. 3. The nature of the, albeit small, variation in the catalytic characteristics of the anti-phosphate IgG (increase in both kcat, the catalytic rate constant calculated as V/2[IgG] and kcat/Km, the apparent second-order rate constant for the overall catalysed conversion of substrate to products, with increase in Km suggests simultaneous improvement in transition state binding and deterioration in substrate binding as predicted from immunogen design and the postulated general mechanistic basis of antibody catalysis. 4. This interpretation is supported by the difference in the values of the dissociation constant Ki for the competitive inhibition by the transition-state analogue 4-methylphenyl 4-nitrophenyl hydrogen phosphate of reactions catalysed by two representative anti-phosphate IgG samples: for the catalysis with Km = 4.5 microM, Ki = 9 nM and for that with Km = 1.3 microM, Ki = 80 nM.(ABSTRACT TRUNCATED AT 400 WORDS)

Polyclonal antibody-catalysed amide hydrolysis.

1. The activated amide (4-nitroanilide), N-(4-nitrophenyl) N'-butyl-1,4-phenylenediacetamide (III) was synthesized. 2. A polyclonal antibody preparation (PCA 270-29) was elicited in a multigeneration cross-bred sheep (no. 270) and isolated 29 weeks into the immunization schedule by procedures described previously for PCA 270-22 [Gallacher, Jackson, Searcey, Badman, Goel, Topham, Mellor & Brocklehurst (1991) Biochem J. 271, 871-881]. These involved the use of an amide conjugate bonded through the carboxy group of 4-nitrophenyl 4'-carboxymethylphenyl phosphate and an amino group of keyhole-limpet haemocyanin as the immunogen. 3. PCA 270-29 was shown to catalyse the hydrolysis of both the carbonate ester substrate 4-nitrophenyl 4'-(3-aza-2-oxoheptyl)phenyl carbonate (I) and the amide substrate (III). Both catalyses obeyed the Michaelis-Menten equation with the following values of the parameters at 25 degrees C: for the hydrolysis of (I) at pH 8.0, Km = 3.96 +/- 0.28 microM and k(cat.) = 0.135 +/- 0.004 s-1 (k(non-cat.) = 1.99 x 10(-4) s-1); for the hydrolysis of (III) at pH 9.0, Km = 5.4 +/- 1.4 microM and k(cat.) = (5.95 +/- 0.75) x 10(-5) s-1 (k(non-cat.) = approx. 2 x 10(-7) s-1). 4. The finding that PCA 270-29 is almost equally effective as a catalyst for the hydrolysis of the amide (III) as for that of the carbonate ester (I) when allowance is made for the different intrinsic reactivities of the two types of substrate is discussed. The catalytic characteristics of PCA 270-29, the first example of a polyclonal catalytic antibody preparation shown to catalyse the hydrolysis of an amide and the first example of an antibody preparation (monoclonal or polyclonal) with such catalytic character to be produced by use of a phosphate immunogen, are compared with those of the small number of other antibody-mediated hydrolyses of amides in the literature.