The color of urine: then and now-a comprehensive review of the literature with emphasis on intracytoplasmic pigments encountered in urinary cytology.

The color of urine, once considered by uroscopists to give the most important clues to the diagnosis, still can provide some diagnostic clues in modern medicine. Pigmented cells are an uncommon and surprising find in urine cytology and can at the same time provide important diagnostic clues or represent a dangerous pitfall. We present a review of the significance of pigmented cells in urine cytology. The presence of intracellular pigment granules; their color, size, shape, and variation in size and shape; as well as their staining reactions with special stains can provide useful diagnostic insight, especially when interpreted in the cytologic context (type of pigmented cell and its degree of atypicality) and patient's clinical context. The main differential diagnosis of cytoplasmic pigmented granules includes hemosiderin, lipofuscin, and melanin, each having a different pathogenesis and significance. The goal of this paper is to describe the morphological, histochemical, and ultrastructural characteristics of the pigments seen in urinary cytology, and to review the benign and malignant conditions associated with them.

Metabolic profile of anhydrosafflor yellow B in rats by ultra-fast liquid chromatography/quadrupole time-of-flight mass spectrometry.

Anhydrosafflor yellow B (AHSYB) is one of the major active water-soluble pigments from Carthamus tinctorius, which has been found to inhibit ADP-induced platelet aggregation and possess significant antioxidant activity. However, the metabolic fate of AHSYB in vivo remains unknown. In order to explore whether AHSYB is extensively metabolized, the metabolites of AHSYB in plasma, urine, bile, and feces samples after intravenous administration to the rats were investigated by ultra-fast liquid chromatography/quadrupole time-of-flight mass spectrometry (UFLC/Qq-TOF-MS/MS) combined with Metabolitepilot™ software. In total, AHSYB and 22 metabolites including both phase I and phase II metabolism processes were found and tentatively identified from the bio-samples. The metabolic pathways were involved in oxidation, reduction, hydroxylation, methylation, dimethylation, O-acetylation, hydrolyzation, sulfation, glucuronidaton, glutathionation and combination with glucose. The results showed that the renal and biliary routes play an important role in the clearance and excretion of AHSYB as well as hepatocyte metabolism. All of these results were reported for the first time and would contribute to a further understanding of the in vivo intermediated processes and metabolic mechanism of AHSYB and its analogs.

Hematuria and pigmenturia of horses.

Hematuria and pigmenturia of horses are discussed in this article. Equine urine is normally straw colored. Discolored urine can be caused by contamination with red blood cells, hemoglobin, myoglobin, oxidizing agents normally found in urine, and plant-derived pigments.

Safety of hydroxocobalamin in healthy volunteers in a randomized, placebo-controlled study.

INTRODUCTION: This randomized, double-blind, placebo-controlled, ascending-dose study was conducted in healthy volunteers to evaluate the safety of the investigational cyanide antidote hydroxocobalamin. METHODS: Four ascending dosing groups received intravenous doses of 2.5, 5, 7.5 or 10 g hydroxocobalamin over 7.5 to 30 minutes at a constant infusion rate. Volunteers (n = 136) randomized 3:1 to receive hydroxocobalamin or placebo underwent a 4-day in-house observation after infusion on Day 1 and follow-up visits on Days 8, 15, and 28. RESULTS: The most common drug-related adverse events were asymptomatic and self-limiting chromaturia and reddening of the skin, which are attributed to the red color of hydroxocobalamin. Other adverse events included pustular/papular rash, headache, erythema at the injection site, decrease in lymphocyte percentage, nausea, pruritus, chest discomfort, and dysphagia. Hydroxocobalamin was associated with an increase in blood pressure in some volunteers. Blood pressure changes peaked toward the end of hydroxocobalamin infusion and typically returned to baseline levels by 4 hours postinfusion. Maximum mean changes from baseline in systolic blood pressure ranged from 22.6 to 27.0 mmHg across hydroxocobalamin doses compared with 0.2 to 6.7 mmHg in the corresponding placebo groups. Maximum mean change from baseline in diastolic blood pressure ranged from 14.3 to 25.4 mmHg across hydroxocobalamin doses compared with -3.0 to 3.8 mmHg in the corresponding placebo groups. Two allergic reactions that occurred within minutes after start of the 5- and 10-g hydroxocobalamin infusions were successfully managed with dexamethasone and/or dimethindene maleate. CONCLUSION: Timely intervention for acute cyanide poisoning could entail administration of an antidote in the prehospital setting based on a presumptive diagnosis. Results of this placebo-controlled study in healthy volunteers corroborate previous studies and French postmarketing experience in cyanide-exposed patients in suggesting that the safety profile of hydroxocobalamin is consistent with prehospital or hospital use.

Pharmacokinetics and excretion of hydroxysafflor yellow A, a potent neuroprotective agent from safflower, in rats and dogs.

Studies were conducted to characterize the pharmacokinetics and excretion of hydroxysafflor yellow A (HSYA) in rats and dogs after administration by intravenous injection or infusion. Plasma, urine, feces and bile concentrations of HSYA were measured using five validated mild HPLC methods. Linear pharmacokinetics of HSYA after the intravenous administrations were found at doses ranging from 3 to 24 mg/kg in rats and from 6 to 24 mg/kg in dogs. At a dose of 3 mg/kg, HSYA in urine, feces and bile was determined. For 48 h after dosing, the amount of urinary excretion accounted for 52.6 +/- 17.9 % (range: 31.1 - 78.7%, n = 6) of the dose, and the amount of fecal amount accounted for 8.4 +/- 5.3% (range 1.7 - 16.4%, n = 6) of the dose. Biliary excretion amount accounted for 1.4 +/- 1.0% (range 0.4-2.9%; n = 6) of the dose for 24 h after dosing. Percent plasma protein binding of HSYA ranged from 48.0 to 54.6% at 72 h. In summary, five mild HPLC methods for the determinations of HSYA in rat plasma, urine, feces, bile and dog plasma have been developed and successfully applied to preclinical pharmacokinetics and excretion of HSYA in rats and dogs. The results of excretion studies indicated that HSYA was rapidly excreted as unchanged drug in the urine. In view of previous pharmacological work, the concentration-dependent neuroprotective effect of HSYA in rats was defined.

Influence of pigments and pH of urine on the determination of N-acetyl-beta-D-glucosaminidase activity with 2-methoxy-4-(2'-nitrovinyl)-phenyl-N-acetyl-beta-D-glucosaminide.

The influence of urinary pigments and urine pH on the spectrophotometric determination of N-acetyl-beta-D-glucosaminidase (NAG; EC 3.2.1.30) activity with 2-methoxy-4-(2'-nitrovinyl)-phenyl-N-acetyl-beta-D-glucosaminide as a substrate was studied. The investigation was performed with human and rabbit urine samples. It was found that alkaline urine pH values influenced NAG activity in two ways: 1) NAG activity decreased due to enzyme instability with pH increase, and 2) NAG activity increased because of the contribution of urinary pigments to absorbance of 2-methoxy-4-(2'-nitrovinyl)-phenol (MNP) at 505 nm. It was shown that besides the maximum (I) in the range of 350-360 nm of the absorption spectra of alkaline urine, there was a maximum (II) in the range of 380-460 nm. With the increase of pH, maximum II was shifted toward higher wavelengths and contributed to MNP absorption (5-90%). On the other hand, the maximum of MNP absorption was shifted toward lower wavelengths (495-400 nm) with increasing pH. Two procedures to eliminate the influence of urinary pigments are presented. The justification of applying a correction to the values of NAG activity in human and rabbit urine (a model system for studying the toxic effects of cadmium) was discussed.

Characterization of dark liver pigment observed in rats after subchronic dosing of the beta3-adrenergic receptor agonist LY368842.

Dark liver pigmentation was observed in F344 rats in a subchronic toxicology study after daily dosing of LY368842 glycolate. In addition, green-colored urine was observed in some animals. To identify the source of the pigment and its potential for toxic consequences, the liver pigment was isolated from the liver tissue of rats. The resulting material was a dark brown to black powder that was insoluble in water, organic solvents, or a tissue-solubilizing agent. Several techniques, such as chemical degradation, HPLC, tandem mass spectrometry (LC/MS/MS), (1)H NMR, and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), were employed to characterize the dark liver pigment. Following oxidative degradation of the isolated pigment, degradation products related to LY368842 were identified or tentatively identified using LC/MS/MS. Two degradation products had the same protonated molecular ion at m/z 505, which is 30 amu higher than that of LY368842. The major m/z 505 product has been identified as the indole-2,3-dione oxidative product based on (1)H NMR data and confirmed by an authentic standard. In addition, monohydroxylated product was also identified in the degradation mixture. These degradation products were consistent with the metabolites found in vivo in rats. MALDI-MS analyses of liver and urine pigment both identified a product with a protonated molecular ion at m/z 977, suggesting formation of indirubin-like and indigo-like pigments. The results obtained suggest that the oxidative metabolites of LY368842 played a key role in the formation of the liver and urine pigments.

Beeturia and colonic oxalic acid.

Beeturia is the excretion of red beetroot pigment (betalaine) in urine and faeces. It occurs in about 14% of humans. Betalaine is a redox indicator whose colour is protected by reducing agents. We investigated pigment-decolourizing systems in the intestinal tracts of beeturic and non-beeturic subjects. Betalaine was decolourized by hydrochloric acid, ferric ions and colonic bacteria preparations, but not by pancreatic or mucosal enzymes. In animals, oral betalaine did not produce beeturia, but injection of betalaine into the peritoneum did. Oral betalaine and 1 g oxalic acid produced beeturia in non-beeturic normal humans, but passed into ileostomies without beeturia. Thus, beeturia results from colonic absorption of betalaine. Oxalic acid preserves the red colour to the colon, otherwise it is decolourized in non-beeturic individuals by non-enzymic processes in the stomach and colon.

Haematuria, pigmenturia and proteinuria in exercising horses.

The effects of exercise on urinary excretion of red blood cells, pigments (haemoglobin and myoglobin) and protein were studied in 8 mares performing treadmill exercise at speeds eliciting 40, 60 and 95% of the maximal oxygen consumption (VO2max). Gross haematuria and pigmenturia were observed in all horses during exercise at the 2 higher intensities, while these findings were detected in only one of 8 mares during exercise at 40% of the VO2max. For the remaining 7 mares exercised at 40% of the VO2max, increased urinary excretion of red blood cells (RBCs) and pigments was evident after centrifugation of urine samples and reagent strip analysis of the supernatant fractions. An increase in urine flow (UF) during exercise at 40% of the VO2max may have contributed to the infrequent observation of gross haematuria and pigmenturia during exercise at this intensity. A transient increase in UF following exercise at 60 and 95% of the VO2max resulted in rapid resolution of gross haematuria and pigmenturia, but increased urinary excretion of RBCs and pigments remained evident by reagent strip analysis for up to 60 min following exercise. Mean +/- s.e. urinary protein excretion increased from a resting value of 2.2 +/- 0.2 mg/min to 5.6 +/- 0.9, 14.5 +/- 4.7 and 78.4 +/- 18.6 mg/min after exercise at 40, 60 and 95% of the VO2max, respectively. These results demonstrate that exercise induced haematuria and pigmenturia and post exercise proteinuria are common in horses. Their occurrence is transient and does not appear to be associated with any lasting changes in renal function.

Beeturia and the biological fate of beetroot pigments.

Beeturia, the passage of pink or red urine after the ingestion of beetroot, is said to occur in 10-14% of the population, and is more common in iron deficiency and malabsorption. A specific HPLC assay for betacyanins, the red beetroot pigments, in biological fluids was developed to study the prevalence of this apparent polymorphism in humans, and to investigate its basis in rats. Two major peaks were observed in chromatograms of extracts of unpickled beetroot. They had identical UV absorption spectra (lambda max = 535 nm) by diode array analysis, and mass spectrometry indicated that one (betacyanin 1) was betanin or its epimer and the other (betacyanin 2) a disaccharide of betacyanin 1. In a population of 100 normal subjects the 0-8 h urinary recoveries after an oral dose of 60 mg beetroot extract were 0.06-0.54% for betacyanin 1 and 0.01-0.6% for betacyanin 2. The distributions of these data were skewed but not clearly bimodal by visual inspection or by kernel density analysis. Four subjects produced visibly red urine and had betacyanin recoveries at the upper end of the population range. Studies using in situ isolated perfused rat jejunum and liver preparations indicated a negligible absorption of the pigments after 1 h and no detectable metabolism or biliary secretion. Intact anaesthetized rats given i.v. bolus doses of beetroot extract cleared both betacyanins from plasma at the rate of 3.3 +/- 0.9 (SD) ml min-1 (n = 5). The total urinary recovery of both pigments amounted to 80% of the dose, and their renal clearances approached their plasma clearances. These data suggest that beeturia does not arise from deficiencies in hepatic metabolism or renal excretion of betacyanins. After oral administration of beetroot extract to rats the betacyanin content of the stomach decreased rapidly with time but neither the intestines nor the bile duct were stained visibly red. These findings together with those showing instability of the betacyanins in acid conditions suggest that variability in the biological fate of beetroot pigments may be determined largely by gastric pH and emptying rate.

Characterization of the pigment from homogentisic acid and urine and tissue from an alkaptonuria patient.

When urine samples from alkaptonuria patients are allowed to stand, they turn black, presumably owing to the oxidation of homogentisic acid to a melanin-like substance. We report the characterization of the pigments formed by polymerization of (a) the components in the urine from a patient with alkaptonuria and (b) homogentisic acid. The absorption spectra and electron spin resonance signals of these pigments are similar to those of eumelanins. Irradiation of the pigments with nitroblue tetrazolium caused reduction of the tetrazolium; this was partially inhibited by superoxide dismutase. Irradiation of Ehrlich ascites carcinoma cells with the pigments from homogentisic acid or urine caused cell lysis. Since this lysis was inhibited by catalase, we have concluded that it was mediated by H2O2. A similar pigment was also extracted from the tissue from an alkaptonuria patient. It is suggested that the degeneration of tissue in vivo may be due to the deposition of melanin-like pigments in the tissues, probably in combination with metal ions.

The infant with a reddish diaper.

The infant with a reddish diaper presents a diagnostic challenge to the primary care physician. As described by Dr Baumgardner, the cause may be benign, but more ominous disorders must be ruled out.