Pyroluria: Fact or Fiction?

Objective: The term "Mauve factor" (pyrroluria) dates back to 1958 when Dr. Abram Hoffer defined the condition as elevated levels of pyrroles in the urine, currently called hydroxyhemepyrrolin-2-one (HPL). It was suggested that the raised pyrrole levels lead to depletions in zinc and vitamin B6, which, in turn, were hypothesized to result in a range of psychiatric disorders, such as schizophrenia, anxiety, and depression. Treatment implications are supplementation with zinc and B6. This article aimed to review the scientific literature associating pyrroluria with psychiatric symptoms, explore the validity of HPL testing, explore the role of nutrients as treatment options for pyrroluria, and discuss future research directions. Methods: A PRISMA review was conducted using search results from electronic databases PubMed, MEDLINE, PsycINFO, EMBASE from inception to February 2020 using the following keywords: hydroxyhemepyryrrolin (HPL), kryptopyrrole (KP), mauve factor, pyroluria, pyrroluria, monopyrroles. Article reference lists were also scanned and included where relevant. Results: Seventy-three articles were identified of which only three studies identified significantly higher HPL levels in a psychiatric population compared with controls, and there were no placebo-controlled treatment trials directed at pyrroluria. The other 13 clinical studies either showed no association or did not provide adequate data to show group differences in HPL levels. Despite an extensive history of practitioners diagnosing and treating a wide variety of mental health conditions associated with pyrroluria as well as clinical observations of elevated HPL being associated with psychiatric disorders, there was no clear research that showed the following: (1) elevated HPL is robustly associated with increased mental health symptoms, (2) elevated HPL in urine is associated with increased urine excretion of zinc and B6, and (3) high-dose zinc and B6 are an efficacious treatment for mental health problems associated with elevated HPL. Conclusions: Elevated HPL is a clinically observed, but poorly researched biomarker with unclear associations with mental disorders. Based on current evidence, HPL testing is not recommended as a screening or treatment tool. Further research is required in the following areas: establishment of which specific clinical populations exhibit elevated HPL, validation of the chemistry and validity of testing, and controlled trials to establish efficacy of high-dose zinc and B6 as treatment of elevated pyrroles.

Assessment of exposure of metabolites in preclinical species and humans at steady state from the single-dose radiolabeled absorption, distribution, metabolism, and excretion studies: a case study.

The exposure of a drug candidate and its metabolites in humans and preclinical species during drug development needs to be determined to ensure that the safety of drug-related components in humans is adequately assessed in the standard toxicology studies. The in vivo radiolabeled studies in preclinical species and human volunteers provide the total fate of the drug-derived radioactivity including the relative abundance of metabolites. Here, we describe how the single-dose radiolabeled human studies could provide the exposure of circulating metabolites at steady state using a case study of an extensively metabolized drug, lixivaptan. After an oral dose of [(14)C]lixivaptan to humans, a total of nine metabolites were detected in the systemic circulation; eight of them exceeded 10% of the parent exposure (2-41% of total radioactivity). The plasma samples were profiled for all subjects at each time point by high-performance liquid chromatography, and metabolites were quantified using a radioactive detector. On the basis of single-dose area under the concentration-time curve (AUC) values, exposure of six human metabolites was greater at least in one preclinical species used in toxicology evaluation. On the basis of the t(1/2) of lixivaptan and two major metabolites from a single dose in humans, their AUC and C(max) values were simulated at the steady state. The simulated exposure (C(max) and AUC) values of parent drug and the two most abundant metabolites were similar to those from a 7-day clinical study obtained using a validated liquid chromatography-mass spectrometry assay, suggesting that a well designed single-dose radiolabeled human study can help in addressing the metabolites in safety testing-related issues.

A nonclinical safety and pharmacokinetic evaluation of N6022: a first-in-class S-nitrosoglutathione reductase inhibitor for the treatment of asthma.

S-nitrosoglutathione reductase is the primary enzyme responsible for the metabolism of S-nitrosoglutathione (GSNO), the body's main source of bioavailable nitric oxide. Through its catabolic activity, GSNO reductase (GSNOR) plays a central role in regulating endogenous S-nitrosothiol levels and protein S-nitrosation-based signaling. By inhibiting GSNOR, we aim to increase pulmonary GSNO and induce bronchodilation while reducing inflammation in lung diseases such as asthma. To support the clinical development of N6022, a first-in-class GSNOR inhibitor, a 14-day toxicology study was conducted. Sprague-Dawley rats were given 2, 10 or 50 mg/kg/day N6022 via IV administration. N6022 was well tolerated at all doses and no biologically significant adverse findings were noted in the study up to 10 mg/kg/day. N6022-related study findings were limited to the high dose group. One male rat had mild hepatocellular necrosis with accompanying increases in ALT and AST and several male animals had histological lung assessments with a slight increase in foreign body granulomas. Systemic exposure was greater in males than females and saturation of plasma clearance was observed in both sexes in the high dose group. Liver was identified as the major organ of elimination. Mechanistic studies showed dose-dependent effects on the integrity of a rat hepatoma cell line.

Discerning the Mauve factor, Part 2.

"Mauve Factor" was once mistaken for kryptopyrrole but is the hydroxylactam of hemopyrrole, hydroxyhemopyrrolin-2-one (HPL). Treatment with nutrients--particularly vitamin B6 and zinc--reduces urinary excretion of HPL and improves diverse neurobehavioral symptoms in subjects with elevated urinary HPL. Heightened HPL excretion classically associates with emotional stress, which in turn is known to associate with oxidative stress. For this review, markers for nutritional status and for oxidative stress were examined in relationship to urinary HPL. In cohorts with mixed diagnoses, 24-hour urinary HPL correlated negatively with vitamin B6 activity and zinc concentration in red cells (P < .0001). Above-normal HPL excretion corresponded to subnormal vitamin B6 activity and subnormal zinc with remarkable consistency. HPL correlated inversely with plasma GSH and red-cell catalase, and correlated directly with plasma nitric oxide (P < .0001). Thus, besides implying proportionate needs for vitamin B6 and zinc, HPL is a promising biomarker for oxidative stress. HPL is known to cause non-erythroid heme depression, which lowers zinc, increases nitric oxide, and increases oxidative stress. Administration of prednisone reportedly provoked HPL excretion in animals. Since adrenocorticoid (and catecholamine) stress hormones mediate intestinal permeability, urinary HPL was examined in relationship to urinary indicans, presumptive marker for intestinal permeability. Urinary HPL associated with higher levels ofindicans (P < .0001). Antibiotics reportedly reduce HPL in urine, suggesting an enterobic role in production. Potentially, gut is reservoir for HPL or its precursor, and stress-related changes in intestinal permeability mediate systemic and urinary concentrations.

Discerning the Mauve Factor, Part 1.

"Mauve Factor" was once mistaken for kryptopyrrole but is the hydroxylactam of hemopyrrole, hydroxyhemopyrrolin-2-one (HPL). Treatment with nutrients--particularly vitamin B6 and zinc--reduces urinary excretion of HPL and improves diverse neurobehavioral symptoms in subjects with elevated urinary HPL. Heightened HPL excretion classically associates with emotional stress, which in turn is known to associate with oxidative stress. For this review, markers for nutritional status and for oxidative stress were examined in relationship to urinary HPL. In cohorts with mixed diagnoses, 24-hour urinary HPL correlated negatively with vitamin B6 activity and zinc concentration in red cells (P < .0001). Above-normal HPL excretion corresponded to subnormal vitamin B6 activity and subnormal zinc with remarkable consistency. HPL correlated inversely with plasma glutathione and red-cell catalase, and correlated directly with plasma nitric oxide (P < .0001). Thus, besides implying proportionate needs for vitamin B6 and zinc, HPL is a promising biomarker for oxidative stress. HPL is known to cause non-erythroid heme depression, which lowers zinc, increases nitric oxide, and increases oxidative stress. Administration of prednisone reportedly provoked HPL excretion in animals. Since adrenocorticoid (and catecholamine) stress hormones mediate intestinal permeability, urinary HPL examined in relationship to urinary indicans, presumptive marker for intestinal permeability. Urinary HPL associated with higher levels of indicans (P < .0001). Antibiotics reportedly reduce HPL in urine, suggesting an enterobic role in production. Potentially, gut is a reservoir for HPL or its precursor, and stress-related changes in intestinal permeability mediate systemic and urinary concentrations.

[No relationship between so-called 'haemopyrrollactamuria' and fatigue in secondary-school students]. / Geen verband tussen zogeheten 'hemopyrrollactamurie' en vermoeidheid bij gymnasiasten.

OBJECTIVE: To determine the relationship between urinary haemopyrrollactam (HPL) and subjective fatigue in secondary-school students. DESIGN: Cross-sectional and descriptive. METHOD: Urine samples were collected from 75 secondary-school students (43 boys and 32 girls, aged 16-18 years) in Nijmegen, the Netherlands. The samples were tested for HPL complex at the Centre for Environmental Medicine (Klinisch Ecologisch Allergie Centrum; KEAC) in Weert. Students also completed the 'Abbreviated fatigue questionnaire'. A score of 21 points was considered the threshold for fatigue. RESULTS: Of the 75 participants, 22 (29%) had increased HPL complex excretion, including 15 boys (35%) and 7 girls (22%). Overall, 19 (25%) had a fatigue score > 21: 16 boys (37%) and 3 girls (9%). There was no statistical relationship between increased HPL complex excretion and fatigue score. CONCLUSION: These findings do not support a relationship between fatigue and increased urinary HPL complex excretion.

Study of a novel indolin-2-ketone compound Z24 induced hepatotoxicity by NMR-spectroscopy-based metabonomics of rat urine, blood plasma, and liver extracts.

Antiangiogenic compound has been believed to be an ideal drug in the current cancer biological therapy, but the angiogenesis inhibitors suffer setback for unknown toxicity now. A novel synthetic indolin-s-ketone small molecular compound, 3Z-3-[((1)H-pyrrol-2-yl)-methylidene]-1-(1-piperidinylmethyl)-1,3-2H-indol-2-one (Z24) can inhibit angiogenesis in new blood vessels. The hepatotoxicity effects of Z24 oral administration (dosed at 60, 130 and 200 mg/kg) have been investigated in female Wistar rats by using metabonomic analysis of (1)H NMR spectra of urine, plasma and liver extracts, as well as by clinical chemistry analysis, liver histopathology and electron micrographs examination. The (1)H NMR spectra of the biofluids were analyzed visually and via pattern recognition by using principal component analysis. The metabonomic trajectory analysis on the time-related hepatotoxicity of Z24 was carried out based on the (1)H NMR spectra of urine samples, which were collected daily predose and postdose over an 8-day period. Urinary excretion of citrate, lactate, 2-oxo-glutarate and succinate increased following Z24 dosing. Increased plasma levels of lactate, TMAO and lipid were observed, with concomitant decrease in the level of glucose and phosphatidylcholine. Metabolic profiling on aqueous soluble extracts of liver tissues with the high dose level of Z24 showed an increase in lactate and glutamine, together with a decrease in glucose, glycogen and choline. On the other hand, studies on lipid soluble extracts of liver tissues with the high dose level of Z24 showed increased level in lipid triglycerides and decreased level in unsaturated fatty acids and phosphatidylcholine. Moreover, the most notable effect of Z24 on the metabolism was the reduction in the urinary levels of creatinine and TMAO and the increase in acetate, citrate, succinate and 2-oxo-glutamate with time dependence. The results indicate that in rats Z24 inhibits mitochondrial function through altering the energy and lipid metabolism, which results in the accumulation of free fatty acids and lactate because of the lack of aerobic respiration. These data show that the metabonomic approach represents a promising new technology for the toxicological mechanism study.

Glucuronidation of statins in animals and humans: a novel mechanism of statin lactonization.

The active forms of all marketed hydroxymethylglutaryl (HMG)-CoA reductase inhibitors share a common dihydroxy heptanoic or heptenoic acid side chain. In this study, we present evidence for the formation of acyl glucuronide conjugates of the hydroxy acid forms of simvastatin (SVA), atorvastatin (AVA), and cerivastatin (CVA) in rat, dog, and human liver preparations in vitro and for the excretion of the acyl glucuronide of SVA in dog bile and urine. Upon incubation of each statin (SVA, CVA or AVA) with liver microsomal preparations supplemented with UDP-glucuronic acid, two major products were detected. Based on analysis by high-pressure liquid chromatography, UV spectroscopy, and/or liquid chromatography (LC)-mass spectrometry analysis, these metabolites were identified as a glucuronide conjugate of the hydroxy acid form of the statin and the corresponding delta-lactone. By means of an LC-NMR technique, the glucuronide structure was established to be a 1-O-acyl-beta-D-glucuronide conjugate of the statin acid. The formation of statin glucuronide and statin lactone in human liver microsomes exhibited modest intersubject variability (3- to 6-fold; n = 10). Studies with expressed UDP glucuronosyltransferases (UGTs) revealed that both UGT1A1 and UGT1A3 were capable of forming the glucuronide conjugates and the corresponding lactones for all three statins. Kinetic studies of statin glucuronidation and lactonization in liver microsomes revealed marked species differences in intrinsic clearance (CL(int)) values for SVA (but not for AVA or CVA), with the highest CL(int) observed in dogs, followed by rats and humans. Of the statins studied, SVA underwent glucuronidation and lactonization in human liver microsomes, with the lowest CL(int) (0.4 microl/min/mg of protein for SVA versus approximately 3 microl/min/mg of protein for AVA and CVA). Consistent with the present in vitro findings, substantial levels of the glucuronide conjugate (approximately 20% of dose) and the lactone form of SVA [simvastatin (SV); approximately 10% of dose] were detected in bile following i.v. administration of [(14)C]SVA to dogs. The acyl glucuronide conjugate of SVA, upon isolation from an in vitro incubation, underwent spontaneous cyclization to SV. Since the rate of this lactonization was high under conditions of physiological pH, the present results suggest that the statin lactones detected previously in bile and/or plasma following administration of SVA to animals or of AVA or CVA to animals and humans, might originate, at least in part, from the corresponding acyl glucuronide conjugates. Thus, acyl glucuronide formation, which seems to be a common metabolic pathway for the hydroxy acid forms of statins, may play an important, albeit previously unrecognized, role in the conversion of active HMG-CoA reductase inhibitors to their latent delta-lactone forms.

Evidence of zinc protection against 2,5-hexanedione neurotoxicity: correlation of neurobehavioral testing with biomarkers of excretion.

Risk prevention of human exposure against n-hexane neurotoxicity is relevant towards the protective measures to be proposed in occupational toxicology. Metabolic studies have identified 2,5-hexanedione (2,5-HD) as the main neurotoxic metabolite of n-hexane, which reacts with amino groups of lysine in axonal neurofilaments forming 2,5-dimethylpyrrole adducts, which are responsible for n-hexane neurotoxicity. In the present study, we have investigated the interaction of zinc with 2,5-HD, by correlating the decrease of pyrrole derivatives excretion with changes of neurobehavioral effects. Two subchronic experiments (11 and 8 weeks of exposure) were performed in Wistar rats exposed to different doses of 2,5-HD (200, 400 mg/kg per day) and to the mixture of 2,5-HD + zinc acetate (200 + 300 mg/kg per day) and (400 + 500 mg/kg per day). The results obtained show a significant increase in the excretion of pyrroles in the groups exposed to 2,5-HD alone as compared to controls, and a significant decrease in the excretion of pyrrole derivatives in the groups of rats co-exposed to 2,5-HD + zinc acetate when compared to the rats exposed to 2,5-HD alone. These biochemical changes were immediately evident after the first day of exposure. Simultaneously, neurobehavioral testing (rearing and ambulation in open field) was performed weekly in the same groups of rats. The results demonstrated a significant decrease in neurobehavioral dysfunction in rats co-exposed to 2,5-HD and zinc acetate. At the end of the exposure period, pyrroles levels returned to control values progressively, and the recovery of the neurotoxic effects was gradually established depending on the dose of exposure. The results suggest that zinc is a potential chemo-protector against 2,5-HD neurotoxicity which was identified by neurobehavioral testing. Moreover, pyrrole derivatives are good predictive biochemical biomarkers of 2,5-HD exposure and could be used as a complementary tool to characterize its neurotoxic effects.

Megavitamins and learning disorders: a controlled double-blind experiment.

The study investigated the effectiveness of large amounts of ascorbic acid, niacinamide, calcium pantothenate, and pyridoxine when added to a low carbohydrate-high protein diet with 20 learning disabled children. After a double-blind, 6-month period of treatment, the addition of vitamins to the diet failed to produce significant improvements when compared to the diet alone on a variety of intellectual, school achievement, perceptual, and behavioral, measures. Regardless of their group assignment, 18 children showed improvements on a parent-administered behavior checklist. However, without a diet-placebo control group, these gains may have been produced by parental enthusiasm or the children's maturation rather than dietary control. The children's urinary excretion of kryptopyrrole was unrelated to whether or not they showed pre-, post-test gains and, therefore, proved to be invalid as a screening test for "vitamin dependent (on pharmacologic doses) learning disorders."