Massive human ingestion of orpiment (arsenic trisulfide).

BACKGROUND: Because the toxicity of arsenic is well known, arsenic-containing compounds have frequently been ingested for suicidal purposes. We report a case of attempted suicide by massive ingestion of arsenic trisulfide, an arsenic mineral of low solubility, which resulted in minimal symptoms. CASE REPORT: An asymptomatic 57-year-old man presented to an Emergency Department 13h after his reported ingestion of approximately 84g of arsenic contained in a mineral specimen of orpiment (arsenic trisulfide) that had been crushed and mixed with an alcoholic beverage and food. His only symptom before presentation was nausea. Physical examination was unremarkable, and diagnostic tests included a normal electrolyte panel, a normal serum lactate, and a normal electrocardiogram. An abdominal radiograph revealed hyper-dense material scattered throughout the large intestine. As per the recommendations of the regional poison center, the patient was managed with whole bowel irrigation with a polyethylene glycol solution, maintenance intravenous hydration, and observation on a telemetry unit. Chelation was not performed. A spot urine specimen collected 12h after admission contained 1490µg of total arsenic per liter (background range<50µg per liter). The patient remained asymptomatic throughout his hospital course. Follow-up studies revealed a diminution in both intra-abdominal radiopacities and urine arsenic concentration. X-ray diffraction analysis of the specimen confirmed its identity as arsenic trisulfide. CONCLUSIONS: Our experience demonstrates that massive ingestion of a poorly soluble inorganic arsenic compound can be successfully managed with gastrointestinal decontamination alone without chelation, provided that the patient remains asymptomatic during close clinical monitoring.

Possible involvement of S-nitrosylation of brain cyclooxygenase-1 in bombesin-induced central activation of adrenomedullary outflow in rats.

We previously reported that both nitric oxide (NO) generated from NO synthase by bombesin and NO generated from SIN-1 (NO donor) activate the brain cyclooxygenase (COX) (COX-1 for bombesin), thereby eliciting the secretion of both catecholamines (CA) from the adrenal medulla by brain thromboxane A(2)-mediated mechanisms in rats. NO exerts its effects via not only soluble guanylate cyclase, but also protein S-nitrosylation, covalent modification of a protein cysteine thiol. In this study, we clarified the central mechanisms involved in the bombesin-induced elevation of plasma CA with regard to the relationship between NO and COX-1 using anesthetized rats. Bombesin (1 nmol/animal, i.c.v.)-induced elevation of plasma CA was attenuated by carboxy-PTIO (NO scavenger) (0.5 and 2.5 µmol/animal, i.c.v.), but was not influenced by ODQ (soluble guanylate cyclase inhibitor) (100 and 300 nmol/animal, i.c.v.). The bombesin-induced response was effectively reduced by dithiothreitol (thiol-reducing reagent) (0.4 and 1.9 µmol/kg/animal, i.c.v.) and by N-ethylmaleimide (thiol-alkylating reagent) (0.5 and 2.4 µmol/kg/animal, i.c.v.). The doses of dithiothreitol also reduced the SIN-1 (1.2 µmol/animal, i.c.v.)-induced elevation of plasma CA, but had no effect on the U-46619 (thromboxane A(2) analog) (100 nmol/animal, i.c.v.)-induced elevation of plasma CA even at higher doses (1.9 and 9.7 µmol/kg/animal, i.c.v.). Immunohistochemical studies demonstrated that the bombesin increased S-nitroso-cysteine-positive cells co-localized with COX-1 in the spinally projecting neurons of the hypothalamic paraventricular nucleus (PVN). Taken together, endogenous NO seems to mediate centrally administered bombesin-induced activation of adrenomedullary outflow at least in part by S-nitrosylation of COX-1 in the spinally projecting PVN neurons in rats.

In vivo labeling of B16 melanoma tumor xenograft with a thiol-reactive gadolinium based MRI contrast agent.

Murine melanoma B16 cells display on the extracellular side of the plasma membrane a large number of reactive protein thiols (exofacial protein thiols, EPTs). These EPTs can be chemically labeled with Gd-DO3A-PDP, a Gd(III)-based MRI contrast agent bearing a 2-pyridinedithio chemical function for the recognition of EPTs. Uptake of gadolinium up to 10(9) Gd atoms per cell can be achieved. The treatment of B16 cells ex vivo with a reducing agent such as tris(2-carboxyethyl)phosphine (TCEP) results in an increase by 850% of available EPTs and an increase by 45% of Gd uptake. Blocking EPTs with N-ethylmaleimide (NEM) caused a decrease by 84% of available EPTs and a decrease by 55% of Gd uptake. The amount of Gd taken up by B16 cells is therefore dependent upon the availability of EPTs, whose actual level in turn changes according to the extracellular redox microenvironment. Then Gd-DO3A-PDP has been assessed for the labeling of tumor cells in vivo on B16.F10 melanoma tumor-bearing mice. Gd-DO3A-PDP (or Gd-DO3A as the control) has been injected directly into the tumor region at a dose level of 0.1 µmol and the signal enhancement in MR images followed over time. The washout kinetics of Gd-DO3A-PDP from tumor is very slow if compared to that of control Gd-DO3A, and 48 h post injection, the gadolinium-enhancement is still clearly visible. Therefore, B16 cells can be labeled ex vivo as well as in vivo according to a common EPTs-dependent route, provided that high levels of the thiol reactive probe can be delivered to the tumor.

N-Ethylmaleimide differentially inhibits substrate uptake by and ligand binding to the noradrenaline transporter.

Using transfected HEK293 cells that express the human (h) noradrenaline transporter (hNAT), we show differential inhibitory effects of the thiol reagent N-ethylmaleimide (NEM) on [(3)H]NA uptake and [(3)H]nisoxetine binding. Irreversible inhibition of uptake by NEM was complete, faster, and occurred at lower concentrations. Furthermore, hNAT ligands (substrates and inhibitors) prevented NEM-induced inhibition of binding but not that of uptake, indicating different underlying mechanisms of inhibition. NEM-induced uptake inhibition was not primarily due to inhibition of the Na(+)/K(+)-ATPase since ouabain caused only partial inhibition. For the first time, we show that NEM at low concentrations causes a rapid and complete depletion of cellular adenosine triphosphate (ATP) not only in HEK293 cells but also in several other eukaryotic cell lines. Thus, while high NEM concentrations alkylate the NAT protein in a ligand-protectable manner, low concentrations inhibit substrate uptake through a loss of the Na(+) and K(+) gradient as a driving force by depleting cellular ATP.

Transendothelial transport of low-density lipoprotein and albumin across the rat peritoneum in vivo: effects of the transcytosis inhibitors NEM and filipin.

This study was performed to investigate the mechanisms responsible for the transport of albumin and low-density lipoprotein (LDL) across capillary walls in vivo. To separate transcytosis from passive, 'porous' transport, we tested the effects of the transcytosis inhibitors N-ethylmaleimide (NEM) and filipin given intraperitoneally on the peritoneal capillary clearance of LDL and albumin in anesthetized rats undergoing peritoneal dialysis. Radiolabeled human albumin or LDL was given intra-arterially, and (51)Cr-EDTA was infused intravenously. A 2-hour peritoneal dialysis dwell was performed using 16 ml of conventional 1.36% glucose-based dialysis fluid. The clearance of LDL and albumin to the dialysate and the peritoneal mass transfer coefficient for (51)Cr-EDTA were assessed. Following intraperitoneal NEM incubations (0.5-5 mM), there were marked increases in the peritoneal transport of albumin and LDL for NEM doses exceeding 1 mM. For lower NEM doses, there were no reductions in clearance. Filipin incubations (0.2-4 microg/ml) did not affect the clearance of either macromolecule. In conclusion, neither NEM nor filipin caused reductions in albumin or LDL clearance across the peritoneal capillaries. The present data clearly show that NEM and filipin are unsuitable as transcytosis inhibitors in vivo.

Redox manipulation of NMDA receptors in vivo: alteration of acute pain transmission and dynorphin-induced allodynia.

The redox modulatory site of the N-methyl-D-aspartate (NMDA) receptor directly regulates NMDA receptor function. Sulfhydryl reducing agents, such as dithiothreitol (DTT), potentiate NMDA receptor-evoked currents in vitro, whereas oxidizing agents, such as 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), attenuate these currents. In this study, we examined the effect of this redox manipulations on nociceptive spinal cord signaling in mice. Intrathecal (i.t.) administration of DTT (0.1-30 nmol), presumably reducing the NMDA receptor, dose-dependently enhanced NMDA-induced nociceptive behaviors, and this enhancement was blocked by the oxidizing agent, DTNB. Pretreatment with DTT (10 nmol, i.t.) enhanced NMDA-induced tail-flick thermal hyperalgesia and intraplantar formalin-induced nociceptive behaviors. Finally, DTT pretreatment enhanced the long lasting allodynia induced by i.t. administration of dynorphin, whereas post-treatment with DTNB reduced the permanent allodynia induced by dynorphin for 5 days. Potentiation of all four of these NMDA-dependent nociceptive behaviors by DTT suggests that the reduction of the NMDA receptor by endogenous reducing agents may contribute to augmented pain transmission in response to activation by endogenous glutamate. Moreover, blockade of in vivo NMDA receptor reducing agents or oxidation of the NMDA receptor redox site may prove therapeutically useful in the treatment of chronic pain.

Transport properties of 3'-azido-3'-deoxythymidine and 2',3'-dideoxyinosine in the rat choroid plexus.

Transport properties of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (DDI) were characterized in the isolated rat choroid plexus. AZT and DDI competitively inhibited the active transport of [3H]benzylpenicillin, a prototypic organic anion, with Ki values of 85.4 +/- 13.1 and 155 +/- 22 microM, respectively. Accumulation of [3H]DDI was against an electrochemical potential via a saturable process (K(m) = 29.7 +/- 4.9 microM, Vmax = 13.5 +/- 2.4 pmol min-1/microL tissue) that was inhibited by metabolic inhibitors (carbonylcyanide p-trifluoromethoxyphenylhydrazone, 10 microM, and rotenone, 30 microM) and sulphydryl reagents (p-chloromercuribenzoic acid, 100 microM, and p-chloromercuribenzenesulphonic acid, 100 microM), but did not require an inwardly directed Na+ gradient. Accumulation of [3H]DDI was inhibited by benzylpenicillin and AZT in a dose-dependent manner, with IC50 values of 91.6 +/- 28.9 and 294 +/- 84 microM, respectively. In contrast, no significant accumulation of [3H]AZT was observed. These results suggest that DDI is transported, at least in part, by the transport system for organic anions located on the rat choroid plexus, whereas AZT is recognized, but not transported by this system.

Glutathione peroxidase and catalase modulate the genotoxicity of arsenite.

The X-ray hypersensitive Chinese hamster ovary (CHO) cells, xrs-5, are also more sensitive to sodium arsenite in terms of cell growth and micronucleus induction than CHO-K1 cells. Since reactive oxygen species are suggested to be involved in arsenic toxicity, we have measured antioxidant mechanisms in xrs-5 as well as CHO-K1 cells. There were no apparent differences in the activities of superoxide dismutase, glutathione S-transferase, glutathione reductase, and the levels of glutathione between xrs-5 and CHO-K1 cells. However, the activities of glutathione peroxidase and catalase were 5.4- and 5.8-fold lower, respectively, in xrs-5 cells. The addition of catalase or glutathione peroxidase to cultures reduced the arsenite-induced micronuclei in xrs-5 cells. Whereas, simultaneous treatment with mercaptosuccinate, an inhibitor of glutathione peroxidase, and 3-aminotriazole, an inhibitor of catalase, synergistically increased the arsenite-induced micronuclei. These results suggest that both catalase and glutathione peroxidase are involved in defense against arsenite genotoxicity. The xrs-6 cells, another line of x-ray hypersensitive CHO cells, which had 1.6-fold higher catalase activity and 2.5-fold higher glutathione peroxidase activity than xrs-5 cells, were also more sensitive than CHO-K1 cells but were less sensitive than xrs-5 cells to cell growth inhibition of arsenite. Moreover, a 1.6-fold increase of glutathione peroxidase activity by selenite adaptation effectively removed the arsenite-induced micronuclei in CHO-K1 cells. These results suggest that glutathione peroxidase is more important than catalase in defending against arsenite toxicity. Our results also suggest that increasing the intracellular antioxidant level may have preventive or therapeutic effects in arsenic poisoning.

New ulcerative colitis model induced by sulfhydryl blockers in rats and the effects of antiinflammatory drugs on the colitis.

We tried to produce a new ulcerative colitis model in rats by topical administration of sulfhydryl blockers. After male SD rats were fasted for 24 hr, 100 microliters of 3% N-ethylmaleimide (NEM) or iodoacetamide (IA) was introduced into the colon via a Nelaton's catheter. Both NEM and IA caused severe diarrhea with rectal bleeding and decreased body weight for about 7 days. At autopsy, adhesions and dilatation of the colon and severe mucosal lesions were observed. Both the weight and myeloperoxidase activity of the colon increased markedly. Maximum changes were observed within 1-3 days followed by gradual recovery, but even on day 21, some abnormalities were still observed. The ulceration and inflammation of the colon were confirmed by histological studies. Antiinflammatory drugs such as indomethacin inhibited the inflammation of the colon by NEM, but aggravated the ulceration. These results revealed that sulfhydryl blockers instilled into the colon caused ulcerative colitis in the rat. This model may be useful in studies on the pathogenesis of ulcerative colitis and the evaluation of drugs for therapy. Furthermore, it was suggested that antiinflammatory drugs may delay the healing of colonic ulcers.

Enhanced susceptibility of follicle-stimulating-hormone-deprived infertile bonnet monkey (Macaca radiata) spermatozoa to dithiothreitol-induced DNA decondensation in situ.

Immunoneutralization of endogenous follicle-stimulating hormone (FSH) of adult male monkeys leads to oligospermia and infertility despite unchanged testosterone levels. The inability of these monkeys to impregnate despite repeated exposures to cycling females appeared to be due to abnormal alterations in the kinetics of germ cell transformations and deficient spermiogenesis. Here we investigated the stability of sperm chromatin in oFSH-immunized monkeys as a marker for spermiogenesis. The susceptibility of spermatozoa to in vitro decondensation induced by dithiothreitol (DTT, 0.05-50 mM) was studied by measuring the nuclear fluorescence of DTT-treated, ethidium bromide (EB)-stained sperm using flow cytometry. Changes in sperm morphology and binding of thiol-specific 14C-iodoacetamide (14C-IA) were also monitored under the same conditions. Sperm from the immunized monkeys decondensed at a lower concentration of DTT, bound more EB, and decondensed more extensively than those from control animals. The difference was apparent in sperm from all regions of the epididymis. Immunized monkey sperm also bound significantly more 14C-IA at all concentrations of DTT. Overall, the effective concentration of DTT required to elicit 50% of maximal decondensation (ED50) of epididymal and ejaculated sperm was significantly lower for the immunized monkeys than even the caput sperm of controls. These results suggest that FSH deprivation in monkeys results in production of sperm with limited potential for disulfide formation and reduced chromatin stability.

Survival in patients with amyotrophic lateral sclerosis, treated with an array of antioxidants.

Between 1983 and 1988 we treated 36 patients with sporadic amyotrophic lateral sclerosis (ALS) by an array of antioxidants and added other drugs to the regimen whenever a patient reported deterioration. Our customary prescription sequence was N-acetylcysteine (NAC); vitamins C and E; N-acetylmethionine (NAM); and dithiothreitol (DTT) or its isomer dithioerythritol (DTE). Patients with a history of heavy exposure to metal were also given meso 2,3-dimercaptosuccinic acid (DMSA). NAC, NAM, DTT, and DTE were administered by subcutaneous injection or by mouth or by both routes, the other vitamins and DMSA by mouth alone. The hospital pharmacy supplied NAC and NAM injections fluid as 100 ml bottles of 5.0 and 5.85% solutions, respectively. DTT was delivered in special double-walled capsules of 200 mg. DTT/DTE injection fluid was added to the NAC and NAM bottles, the final DTT/DTE concentrations never exceeding 0.5%. DMSA was provided in 250 mg capsules. All of the 36 patients used NAC and DTT/DTE; 29 also used vitamins C and E; 21 also used NAM; and 7 also used DMSA, DMSA, NAM, vitamins C and E were tolerated well. In many patients, DTT, DTE, NAC and NAM induced pain, redness and swelling at the injection sites in that order of decreasing frequency. DTT and DTE did often and NAC did sometimes cause gastric pain, nausea and other abdominal discomfort. Comparison of survival in the treated group and in a cohort of untreated historical controls, disclosed a median survival of 3.4 years (95% confidence interval: 3.0-4.2) in the treated and of 2.8 (95% confidence interval 2.2-3.1) years in the control patients. This difference may be explained by self-selection of our highly motivated treated group and by its initial survival of diagnosis for an average of 8.5 months before onset of treatment. We conclude that antioxidants neither seem to harm ALS patients, nor do they seem to prolong survival.

Sulphydryl blocker induced gastric damage is ameliorated by scavenging of free radicals.

BACKGROUND: Sulphydryl compounds and nitric oxide are essential in maintaining gastric mucosal integrity. AIMS: To characterise the gastric damage induced by a sulphydryl blocker, to evaluate the role of nitric oxide in its pathogenesis, and to reveal its possible prevention by scavenging of free radicals. METHODS: Gastritis was induced in rats by addition of iodoacetamide (0.1%) to the drinking water, with and without daily intragastric administration of TEMPOL. After death, the stomach was resected, washed, lesion area assessed, and mucosal inflammatory mediators, myeloperoxidase and nitric oxide synthase activities were determined. RESULTS: Administration of iodoacetamide induced gastric mucosal erosions present for up to two weeks. Myeloperoxidase activity was increased for up to seven days and nitric oxide synthase activity was significantly decreased for up to 14 days. Treatment for seven days with the free radical scavenger, TEMPOL, decreased by 68% the damage induced by iodoacetamide. CONCLUSIONS: Gastric damage induced by iodoacetamide, a sulphydryl alkylator, accompanied by inhibition of nitric oxide synthase activity shows the important contribution of sulphydryl compounds and nitric oxide to the maintenance of gastric mucosal integrity. Nitric oxide donation and scavenging of free radicals may be a novel approach to prevent gastric damage.

The role of gastric mucosal sulphydryls in the ulcer-protecting effects of cisapride.

The present study was designed to examine the role of endogenous sulphydryls (SHs) in the gastro-protection induced by cisapride (CIS) (10, 25 and 50 mg kg-1 i.p.), a potent benzamide stimulating gastrointestinal motility in mucosal injury induced by 50% v/v ethanol. Results were compared with those of 5-hydroxytryptamine (5-HT) (10mg kg-1). Ethanol mucosal damage was significantly reduced by treatment with CIS and 5-HT. On the contrary, administration of n-ethylmaleimide (NEM) (10 mg kg-1) an SH alkylator, markedly worsened lesion formation and counteracted the protective effect of CIS. Rats pretreated with CIS significantly increased the total sulphydryls as reflected in the non-protein and protein fractions however, 5-HT treatment showed a fall in the non-protein level. The present results suggest that 5-HT-ergic dependent mechanisms have no relation to the gastro-protection afforded by CIS in this experimental model. It is possible that mucosal SHs could be involved.

Retroperfusion studies of the aqueous outflow system. Part 2: Studies in human eyes.

PURPOSE: To extend the retroperfusion technique to allow the delivery of drugs into Schlemm's canal in enucleated human eyes and to use this technique to gain insights into the function of the inner wall of Schlemm's canal. METHODS: Using our previously developed retroperfusion technique, the anterior chamber of enucleated human eyes was held at a small negative pressure (-0.75 mm Hg), and fluid was allowed to flow retrograde from the limbal vessels, through the collector channels, and into Schlemm's canal. In this manner, the sulfhydryl agent N-ethyl maleimide (NEM) or the fixative agent glutaraldehyde was delivered to the inner wall of Schlemm's canal in normal and glaucomatous human eyes. Facility changes caused by retroperfusion were measured and correlated with histologic studies of the inner wall of Schlemm's canal. RESULTS: Retroperfusion effectively delivers fluid from the scleral surface into the lumen of Schlemm's canal. Retroperfusion with vehicle alone does not alter facility or change outflow pathway morphology. Retroperfusion with NEM causes an approximately 35% facility increase and concomitant inner wall openings. Retroperfusion with glutaraldehyde in normal eyes and eyes with primary open-angle glaucoma causes a facility decrease of 53% and 64%, respectively, and localized fixation of the inner wall of Schlemm's canal. The magnitude of the facility changes caused by retroperfusion were similar to those seen using conventional forward perfusion of NEM and glutaraldehyde. CONCLUSIONS: Retroperfusion is a viable technique for the delivery of drugs or other agents into Schlemm's canal in enucleated human eyes. Retroperfusion-induced changes in outflow facility are correlated strongly with morphologically observed alterations in inner wall structure. The majority of outflow resistance is localized to the inner wall of Schlemm's canal or the immediately adjacent 10-microns region of the juxtacanalicular tissue in normal eyes and in eyes with primary open-angle glaucoma. Inner wall giant vacuoles and pores likely persist for sometime, even after fixation at zero or negative pressure.

An attempt to explain interindividual variability in 24-h urinary excretion of inorganic arsenic metabolites by C57 BL/6J mice.

Forty C57 BL/6J mice, injected subcutaneously with 0.5 mg/kg arsenic as sodium arsenite, were examined for 24-h urinary excretion of total arsenic metabolites, creatinine and S-adenosylmethionine (SAM) and for 24-h faecal excretion of arsenic and levels of arsenic in the blood, liver, kidneys, lung, skin, spleen and bone at 24-h post-dose. Total urinary arsenic metabolites were calculated by summing up the inorganic (Asi), monomethylated (MMA) and dimethylated (DMA) derivatives directly measured by selective arsine generation-atomic absorption spectrometry (AG-AAS) or were measured by AG-AAS following complete mineralization. Both sets of results showed interindividual differences varying by as much as 7-fold and correlated with the 24-h urinary excretion of both SAM (r = 0.84 and r = 0.86, respectively) and creatinine (r = 0.82 and r = 0.87, respectively). There was interindividual variability of about a 30-fold range in 24-h faecal excretion of arsenic which correlated inversely with 24-h urinary excretion of arsenic metabolites (r = -0.69) and 24-h urinary excretion of both creatinine (r = -0.70) and SAM (r = -0.67). Body tissue levels of arsenic were low and not related to 24-h urinary excretion of arsenic metabolites, SAM and creatinine. Taken together, the results indicate that differences in the profile of urinary arsenic excretion and in the retention of arsenic in a particular organ do not contribute to interindividual variability in 24-h urinary excretion of arsenic metabolites by C57 BL/6J mice, but that variability in faecal excretion does, at least in part. It is speculated that there is most likely a predominant contribution from a diffuse tissue retention of arsenic or from a third route of arsenic elimination, i.e. respiratory, to this phenomenon in view of the small faecal contribution.

Time-dependent porphyric response in mice subchronically exposed to arsenic.

1. A time-course study was carried out in mice subchronically exposed to As III (as sodium arsenite) or As V (as sodium arsenate), via drinking water, relating the pattern of urinary porphyrin excretion to the renal and hepatic enzyme activities of porphobilinogen deaminase (PBGD), uroporphyrinogen III synthetase (URO III-S), uroporphyrinogen decarboxylase (URO-D) and coproporphyrinogen oxidase (COPRO-O), as well as to the hepatic porphyrin accumulation in the treated animals. 2. A time-dependent, wave-like porphyric response was found in mice exposed to As V, and the increases seen in total urinary porphyrins (at 3 weeks of exposure) corresponded to an increased activity of PBGD and Uro III-S in liver. 3. Significant decreases in renal URO-D and hepatic and renal COPRO-O activities were found in treated mice; these inhibitions were more pronounced in animals exposed to As III. 4. The combination of these enzymic effects may explain the time-dependent porphyric response of mice subchronically exposed to As. Finally, the relative magnitudes of URO-D and COPRO-O inhibitions may determine the pattern of porphyrin concentration observed in urine and tissues. 5. The decrease in renal URO-D activity may help to explain the inversion in the coproporphyrin/uroporphyrin ratio previously reported in humans chronically exposed to As; however, there were differences between the urinary porphyrin profiles found in both species. The possible reasons for the similarities and differences are briefly discussed.

In search of optimum conditions for the measurement of creatine kinase activity: a critical review of nineteen formulations.

We compare the reagent composition recommended by six different groups including three European societies for the determination of creatine kinase activity in serum using the coupled hexokinase/glucose-6-phosphate dehydrogenase (EC 2.7.1.1/1.1.1.49) reactions. Even though discrepancies exist between these methods, there are, nevertheless, major areas of consensus which permit a reasonable extrapolation of an approximate composition for optimum response. We then ascertain how reagents used in thirteen commercial kits differ from these approximated optimum conditions. Except for four companies, all the reagent compositions differ remarkably from the conditions recommended by the six groups.