Green manufacturing of a hypoxanthine enzyme sensor for fish freshness based on modified nitrocellulose surface with chito-oligosaccharide.

Hypoxanthine (Hx), produced by adenosine triphosphate (ATP) metabolism, is a valuable indicator that determines the quality and degradation status of meat products and is also an important biochemical marker to certain diseases such as gout. The rapid emergence of paper-based enzyme biosensors has already revolutionized its on-site determination. But it is still limited by the complex patterning and fabrication, unstable enzyme and uneven coloration. This work aims to develop an eco-friendly method to construct engineered paper microfluidic, which seeks to produce reaction and non-reaction zones without any patterning procedure. Chito-oligosaccharide (COS), derived from shrimp shells, was used to modify nitrocellulose membranes and immobilize xanthine oxidase (XOD) and chromogenic agent of nitro blue tetrazolium chloride (NBT). After modification, micro fluids could converge into the modification area and Hx could be detected by XOD-catalyzed conversion. Due to the positively charged cationic basic properties of COS, the enzyme storage stability and the color homogeneity could be greatly strengthened through the electrostatic attraction between COS and XOD and formazan product. The detection limit (LOD) is 2.30 µM; the linear range is 0.05-0.35 mM; the complete test time can be as short as 5 min. The COS-based biosensor shows high specificity and can be used directly for Hx in complex samples such as fish and shrimp samples, and different broths. This biosensor is eco-friendly, nontechnical, economical and therefore a compelling platform for on-site or home-based detection of food freshness.

Hydrophilic interaction liquid chromatography based method for simultaneous determination of purines and their derivatives in food spices.

This work presents method for separation and quantification of adenine, guanine, xanthine, hypoxanthine, uric acid, and creatinine in food spices using hydrophilic interaction liquid chromatography with UV detection. Optimized conditions allowed separation with mobile phases containing acetonitrile and additives ammonium acetate (90:10, v/v, pH 6.1) or formate (90:10, v/v, pH 3.2). In food spices no uric acid was detected, creatinine (16 ± 2 µg g-1) was found only in instant dried yeast. The highest content of purines was determined in dried yeast (xanthine 110 ± 8 µg g-1, hypoxanthine 441 ± 24 µg g-1, adenine 84 ± 16 µg g-1, guanine 163 ± 12 µg g-1), high in curry, herbal pepper, and chicken seasoning, the lowest concentration was in black pepper (hypoxanthine 12 ± 2 µg g-1, adenine 27 ± 3 µg g-1). To best of our knowledge, no such complementary method and obtained data have been reported so far.

Assessing Meat Freshness via Nanotechnology Biosensors: Is the World Prepared for Lightning-Fast Pace Methods?

In the rapidly evolving field of food science, nanotechnology-based biosensors are one of the most intriguing techniques for tracking meat freshness. Purine derivatives, especially hypoxanthine and xanthine, are important signs of food going bad, especially in meat and meat products. This article compares the analytical performance parameters of traditional biosensor techniques and nanotechnology-based biosensor techniques that can be used to find purine derivatives in meat samples. In the introduction, we discussed the significance of purine metabolisms as analytes in the field of food science. Traditional methods of analysis and biosensors based on nanotechnology were also briefly explained. A comprehensive section of conventional and nanotechnology-based biosensing techniques is covered in detail, along with their analytical performance parameters (selectivity, sensitivity, linearity, and detection limit) in meat samples. Furthermore, the comparison of the methods above was thoroughly explained. In the last part, the pros and cons of the methods and the future of the nanotechnology-based biosensors that have been created are discussed.

The biochemistry of the vitreous humour in estimating the post-mortem interval-a review of the literature, and use in forensic practice in Galicia (northwestern Spain).

The K+ and hypoxanthine (Hx) concentrations of the vitreous humour (VH) rise gradually after death, providing a means of estimating the post-mortem interval (PMI). The correlation between these analytes and the PMI is good since the vitreous chamber is partially isolated from autolytic events occurring elsewhere; the [K +] and [Hx] recorded is thus the result of changes within the eye. The present work provides a systematic review, following PRISMA recommendations, of 36 articles (3 reviews and 33 retrospective cohort studies) discussing the many procedures and regression models that have been developed for improving PMI estimates involving VH analytes. The results of a descriptive study are also provided, highlighting the causes and distribution of mortality as registered in medico-legal autopsies performed in 2019 in Galicia (northwestern Spain), and revealing the use of these PMI estimation methods in real forensic practice. Great heterogeneity was detected in the collection of VH samples, the treatments to which they were subjected before examination, and in their conservation and analysis. A lack of reproducibility in the analytical methods employed to estimate [K +] and [Hx] was noted, as well as an absence of external validation for most of the regression formulae used to determine the PMI from analyte values. The use of methods based on high-performance liquid chromatography, focal electrophoresis, or thermogravimetric/chemometric procedures might solve the problems encountered with traditional analytical techniques, offering reliable results more quickly and effectively (even when samples are contaminated). This study recommends using flexible multiple regression models that combine physical and chemical variables, and that population databases be constructed so that models can be properly validated.

Enzymatic determination of hypoxanthine in fish samples as a freshness indicator using the CUPRAC colorimetric sensor.

Fish consumption is essential for a healthy diet. However, all seafood including fish are susceptible to deterioration unless properly preserved. Controlling the freshness of fresh or packaged fish is a challenging issue for the food industry in terms of human health and shelf life determination. One of the main indicators showing the freshness of fish is undoubtedly the amount of hypoxanthine (Hx). As soon as the organism dies, Hx begins to be released with the cessation of ATP synthesis and shows a gradual increase over time. Therefore, Hx determination is an important indicator in the control of fish freshness. Based on this fact, a colorimetric method for the enzymatic determination of Hx using the CUPRAC (Cupric ion Reducing Antioxidant Capacity) sensor was developed. Uric acid (UA) and H2O2 are enzymatically produced by xanthine oxidase (XOD) from Hx, and both products respond to the CUPRAC reagent to produce the cuprous neocuproine (Cu(I)-Nc) chromophore chelate formed in situ on a Nafion anionic membrane on which the cationic Cu(II)-Nc complex was fixed. Hx was measured at different time intervals in the meat samples taken from sea bass (Dicentrarchus labrax), which was left to stand at room temperature for a time period between 0 and 24 h; the level of spoilage was determined from the coloration of the CUPRAC membrane sensor (via absorbance measurement at 450 nm). It was observed that there was a linear increase in the amount of Hx during the measurement period. The method was optimized for Hx determination, verified with interference analysis and standard additions to real samples, and validated against HPLC. The linear detection range of the developed method for Hx was 2.0-32.0 µM with an LOD of 0.79 µM, and early stages of fish degradation could be detected at several nanomoles of Hx per gram of fish meat. The proposed method was demonstrated to have distinct superiority over many recent colorimetric sensors of fish freshness in regard to its lower LOD for Hx, wider linear range, capability to cope with interferents (including biologically important antioxidants, such as cysteine, reduced glutathione, ascorbic acid, UA and α-tocopherol) and applicability to real samples.

A pattern-free paper enzyme biosensor for one-step detection of fish freshness indicator hypoxanthine with a microfluidic aggregation effect.

In this study, a paper-based enzyme biosensor for hypoxanthine (Hx) was developed, enabling visual and one-step fish freshness detection. Xanthine oxidase and horseradish peroxidase were immobilized on nitrocellulose membranes with 3,3',5,5'-tetramethylbenzidine to output the colour signal. Chitosan oligosaccharide lactate-modified nitrocellulose membranes entrapped the dual-enzyme system and exhibited excellent microfluidic aggregation effect. The developed enzyme biosensor produced a linear response of 0.01-0.16 mmolL-1 with a detection limit of 8.22 µmolL-1, and was selective for Hx with recoveries of 96.13-103.11 % for fish samples. These biosensors were attached directly to the surface of fish samples and the colour was revealed within 3 min. Colour signals can be judged by the naked-eye to distinguish between fresh and spoiled fish samples and analyzed by a smartphone for quantitative analysis. The biosensor shows great potential as a powerful pattern- and reagent-free device for on-site freshness evaluation of fish.

Spectroscopic investigation of faeces with surface-enhanced Raman scattering: a case study with coeliac patients on gluten-free diet.

Surface-enhanced Raman scattering (SERS) spectra of faecal samples can be obtained by adding AuNP to their methanol extracts according to the reported protocol, and display bands that are due to bilirubin-like species but also to xanthine and hypoxanthine, two metabolic products secreted by gut bacteria. A total of 27 faecal samples from three different groups, i.e. coeliac patients (n = 9), coeliac patients on gluten-free diet (n = 10) and a control group (n = 8), were characterized with both SERS spectroscopy and 16S rRNA sequencing analysis. Significant differences are present between SERS spectra of coeliac patients and those on gluten-free diet, with a marked increase in the relative intensity of both xanthine and hypoxanthine for the latter. Interestingly, these differences do not correlate with bacterial composition as derived from 16S rRNA sequencing.

The impact of physiological metabolite levels on serine uptake, synthesis and utilization in cancer cells.

Serine is a non-essential amino acid that is critical for tumour proliferation and depletion of circulating serine results in reduced tumour growth and increased survival in various cancer models. While many cancer cells cultured in a standard tissue culture medium depend on exogenous serine for optimal growth, here we report that these cells are less sensitive to serine/glycine depletion in medium containing physiological levels of metabolites. The lower requirement for exogenous serine under these culture conditions reflects both increased de novo serine synthesis and the use of hypoxanthine (not present in the standard medium) to support purine synthesis. Limiting serine availability leads to increased uptake of extracellular hypoxanthine, sparing available serine for other pathways such as glutathione synthesis. Taken together these results improve our understanding of serine metabolism in physiologically relevant nutrient conditions and allow us to predict interventions that may enhance the therapeutic response to dietary serine/glycine limitation.

Influence of xanthine oxidoreductase inhibitor, topiroxostat, on body weight of diabetic obese mice.

Plasma xanthine oxidoreductase (XOR) activity is high in metabolic disorders such as diabetic mellitus, obesity, or overweight. Thus, this study investigated whether the XOR inhibitor, topiroxostat, affected body weight. Male db/db mice were fed standard diets with or without topiroxostat for 4 weeks. Body weight and food intake were constantly monitored, along with monitoring plasma biochemical markers, including insulin and XOR activity. Additionally, hepatic hypoxanthine and XOR activity were also documented. Single regression analysis was performed to determine the mechanism. Topiroxostat treatment suppressed weight gain relative to the vehicle without any impact on food intake. However, the weight of fat pads and hepatic and muscle triglyceride content did not change. Topiroxostat decreased the plasma uric acid and increased hepatic hypoxanthine in response to the inhibition of XOR activity. Plasma ketone body and free fatty acid were also increased. Moreover, fat weight was weakly associated with plasma XOR activity in the diabetic state and was negatively associated with ketone body by topiroxostat. These results suggested that topiroxostat amplified the burning of lipids and the salvage pathway, resulting in predisposing the body toward catabolism. The inhibition of plasma XOR activity may contribute to weight loss.

Detection of Hypoxanthine from Inosine and Unusual Hydrolysis of Immunosuppressive Drug Azathioprine through the Formation of a Diruthenium(III) System.

Hypoxanthine (hpx) is an important molecule for both biochemistry research and biomedical applications. It is involved in several biological processes associated to energy and purine metabolism and has been proposed as a biomarker for a variety of disease states. Consequently, the discovery and development of systems suitable for the detection of hypoxanthine is pretty appealing in this research field. Thus, we have obtained a stable diruthenium (III) compound in its dehydrated and hydrated forms with formula [{Ru(µ-Cl)(µ-hpx)}2Cl4] (1a) and [{Ru(µ-Cl)(µ-hpx)}2Cl4]·2H2O (1b), respectively. This purine-based diruthenium(III) system was prepared from two very different starting materials, namely, inosine and azathioprine, the latter being an immunosuppressive drug. Remarkably, it was observed that an unusual azathioprine hydrolysis occurs in the presence of ruthenium, thus generating hypoxanthine instead of the expected 6-mercaptopurine antimetabolite, so that the hpx molecule is linked to two ruthenium(III) ions. 1a and 1b were characterized through IR, SEM, powder and single-crystal X-ray Diffraction and Cyclic Voltammetry (CV). The electrochemical studies allowed us to detect the hpx molecule when coordinated to ruthenium in the reported compound. The grade of sensitivity, repeatability and stability reached by this diruthenium system make it potentially useful and could provide a first step to develop new sensor devices suitable to detect hypoxanthine.

Effect of allicin and its mechanism of action in purine removal in turbot.

Low-purine food is not only the focus of gout patients, but also the focus of contemporary green diet development. Fish are usually considered as high-purine foodstuff because of the high nutritional value and high purine content. Therefore, it is necessary to reduce purine content in fish to ensure that they are suitable for patients with hyperuricemia or gout. In this study, the effect of allicin on purine reduction in turbot during cooking was investigated using high-performance liquid chromatography, and the change of xanthine oxidase (XO) activity was also studied. Molecular docking analysis was further performed to elucidate the mechanism of purine reduction by allicin. The results revealed that in the step of soaking, allicin could reduce purine content in fish by slightly enhancing XO activity, promoting hypoxanthine transformation into xanthine. The removal of total purines in experimental and control group reached 70.45% and 57.20%, respectively. Moreover, allicin could change the thermal stability of xanthine by providing an acidic environment, resulting in the rapid decrease of xanthine and hypoxanthine levels by boiling. Thus, this study provides a simple method to decrease purine levels, suggesting a possibility that allicin can function as a purine remover in food.

Circadian movement behaviours and metabolism differences of the Pacific abalone Haliotis discus hannai.

Circadian rhythm is the most important and universal biological rhythm in marine organisms. In this research, the movement behaviour of abalone (Haliotis discus hannai) was continuously monitored under a light cycle of 12 L:12D. It was found that the cumulative movement distance and cumulative movement time of abalone reached was highest from 00:00-03:00 h. The minimum values of maximum movement velocity occurred between 21:00-00:00 h, and a significant circadian cosine rhythm was exhibited during these periods (P < 0.05). Metabolomic analysis of cerebral ganglions of abalone was conducted at 06:00 h (6 M), 14:00 h (14 M), and 22:00 h (22 M) and 380, 385, and 315 metabolites with significant differences were identified in 6 M vs 14 M, 14 M vs 22 M, and 6 M vs 22 M, respectively (P < 0.05). With the alternation of day and night, the expression levels of phosphatidylcholine, 5-HT, N-acetyl-5-hydroxytryptamine, indole-3-acetaldehyde, hypoxanthine, and deoxyinosine declined significantly, while those of Lysophosphatidylcholines (lysoPC) (20: 5 (5Z, 8Z, 11Z, 14Z, 17Z)), lysoPC (22: 4 (7Z, 10Z, 13Z, 16Z)), lysoPC (16: 1 (9Z) / 0: 0), phosphatidylethanolamine (PE) (18: 1 (11Z) 22: 2 (13Z, 16Z)), and guanosine 5'-phosphate rose significantly. These 11 metabolites can be used as differential metabolic markers. These findings not only quantitatively describe the circadian movement behaviours of abalone, but also provide an initial analysis of the circadian mechanism of the physiological metabolic conversion of abalone, which in turn provides guidelines for light control and feeding strategy for use in aquaculture production.

A simple electrochemical approach to fabricate functionalized MWCNT-nanogold decorated PEDOT nanohybrid for simultaneous quantification of uric acid, xanthine and hypoxanthine.

Medical diagnostics and detection of food spoilage require estimation of hypoxanthine (HX), xanthine (XN), and uric acid (UA). A selective sensing platform has been proposed for simultaneous detection of all these species. Functionalized multi-walled carbon nanotube (fMWCNT) stabilized nanogold decorated PEDOT:TOS polymeric nanocomposite (Au-PEDOT-fMWCNT) was synthesized through rapid one-step electropolymerization to enhance conductivity and active surface area by several folds. Electrochemical activities of the proposed sensing platform were analyzed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV), electrochemical impedance spectroscopy (EIS). Analyses through SEM, FESEM and TEM were performed to explore the surface morphology and elemental analysis of the polymeric nanohybrid was investigated by XPS, Raman, FTIR, XRD spectroscopy. Electro-catalysis of UA, XN and HX occurred at low oxidation potentials i.e. 0.082, 0.463 and 0.808 V, respectively in the optimized conditions. The uniquely designed simple, interference free Au-PEDOT-fMWCNT/GCE sensor exhibited high selectivity, good reproducibility, reusability (∼180 times) and stability (∼3 month) with excellent sensitivity of 1.73, 14.31 and 3.82 µA µM-1 cm-2 for UA, XN and HX, respectively. The sensor exhibited linear ranges of detection as 0.1-800, 0.05-175 and 0.1-150 µM with detection limits of 199.3, 24.1 and 90.5 nM for quantification of UA, XN and HX respectively. The performance of the proposed sensor was validated by addition of UA, XN and HX in human serum, urine and fish samples by comparing to those using HPLC. The results indicated good applicability of the proposed sensor for simultaneous detection of UA, XN, HX in real biological fluids.

Low-temperature and long-time heating regimes on non-volatile compound and taste traits of beef assessed by the electronic tongue system.

The influence of temperature-time combinations on non-volatile compound and taste traits of beef semitendinosus muscles tested by the electronic tongue was studied. Single-stage sous-vide at 60 and 70 °C (6 and 12 h), and two-stage sous-vide that sequentially cooked at 45 °C (3 h) and 60 °C (either 3 or 9 h) were compared with traditional cooking at 70 °C (30 min). Umami was better explained in the given model of partial least squares regression than astringency, sourness, saltiness, bitterness, and richness. Sous-vide at 70 °C for 12 h characterized the most umami, likely adenosine-5'-monophosphate (AMP) and guanosine-5'-monophosphate (GMP) as significant contributors. Two-stage sous-vide projected higher histidine, leucine, inosine, and hypoxanthine with the astringent and sour taste significant after 6 and 12 h cooking, respectively. Equivalent umami concentration (EUC) between umami amino acids and umami nucleotides showed a strong relationship to umami taste assessed by the electronic tongue.

A fluorescent biosensor based on catalytic activity of platinum nanoparticles for freshness evaluation of aquatic products.

In this study, a fluorescence biosensor based on the peroxidase mimicking activity of platinum nanoparticles (Pt NPs) was fabricated for rapid detection of hypoxanthine (Hx), which is a sensitive indicator of the freshness of aquatic products. The fluorescence intensity of the sensing system had a linear relationship with the concentration of Hx in the range of 8-2500 µM, and the limit of detection was as low as 2.88 µM (S/N = 3). Moreover, benefiting from the excellent selectivity of the biosensor, Hx content in fish, shrimp and squid samples could be quickly detected with good recovery rates (103.94-109.00%). And the Pt NPs used in the biosensor was reusable, which was proved by the recovery rate was only slightly decreased to 91% after three cycles. In addition to the advantages of facile preparation and low cost, the proposed biosensor will be a promising candidate for rapid and convenient freshness evaluation of aquatic products.

Recent progress in nanomaterial-based electrochemical and optical sensors for hypoxanthine and xanthine. A review.

This review (with 160 ref.) summarizes the progress that has been made in the methods for chemical or biochemical sensing of hypoxanthine and xanthine, which are produced as part of purine metabolism and are precursors of uric acid. An introduction discusses the importance of hypoxanthine and xanthine as analytes due to their significance in the clinical and food science, together with the conventional methods of analysis. A large section covers methods for the electrochemical hypoxanthine and xanthine sensing. It is divided into subsections according to the nanomaterials used including carbon nanomaterials, meal oxide nanoparticles, metal organic frameworks, conductive polymers, and bio-nanocomposites. A further large section covers optical methods for hypoxanthine and xanthine sensing, with subsections on nanomaterials including carbon nanomaterials, nanosheets, nanoclusters, nanoparticles, and their bio-nanocomposites. A concluding section summarizes the current status, addresses current challenges, and discusses future perspectives. Graphical abstractSchematic representation of the hypoxanthine and xanthine electrochemical and optical sensors incorporating various nanomaterials like graphene, carbon nanotubes (CNT), quantum dots (QD), nanoparticles and polymers, which are implemented in clinical and food analysis.

Triethylamine-controlled Cu-BTC frameworks for electrochemical sensing fish freshness.

The simultaneous determination of xanthine (XA) and hypoxanthine (HXA) has been proved to be a feasible approach for the assessment of fish freshness. In this study, copper(II) nitrate and 1,3,5-benzenetricarboxylic acid (H3BTC) were used as precursors to prepare various Cu-BTC frameworks with the addition of various amounts of triethylamine at room temperature. The characterization of X-ray diffraction, Fourier-transform infrared spectroscopy and Raman spectroscopy testified that the obtained materials are Cu-BTC frameworks. However, the amount of triethylamine had significant effects on the morphology, active response area and electron transfer ability of Cu-BTC frameworks. The oxidation behavior of XA and HXA demonstrated that the prepared Cu-BTC frameworks exhibited higher sensing activity, with greatly-enhanced oxidation signals. More importantly, the amount of triethylamine obviously affected the accumulation capacity and signal enhancement ability of Cu-BTCs toward XA and HXA, as confirmed from double potential step chronocoulometry. Based on the triethylamine-tuned signal amplification strategy of Cu-BTC frameworks, a highly-sensitive and simple electrochemical sensing system was developed for the assessment of fish freshness by simultaneous detection of XA and HXA. The developed sensing method was used in practical samples, and the results were validated by high-performance liquid chromatography.

Impaired antioxidant capacity causes a disruption of metabolic homeostasis in sickle erythrocytes.

This study examined particularly relevant redox pathways such as glycolysis, pentose phosphate pathway (PPP), metHb reductase and nucleotide metabolism, in order to better address how sickle cells deal with redox metabolism disruption. We also investigated the generation of specific oxidative lesions, and the levels of an unexplored antioxidant that could act as a candidate biomarker for oxidative status in sickle cell anemia (SCA). We adopted rigorous exclusion criteria to obtain the studied groups, which were composed by 10 subjects without hemoglobinopathies and 10 SCA patients. We confirmed that sickle cells overwhelm the antioxidant defense system, leading to an impaired antioxidant capacity that significantly contributed to the increase in cholesterol oxidation (ChAld) and hemolysis. Among the antioxidants evaluated, ergothioneine levels decreased in SCA (two-fold). We found strong correlations of ergothioneine levels with other erythrocyte metabolism markers, suggesting its use as an antioxidant therapy alternative for SCA treatment. Moreover, we found higher activities of MetHb reductase, AChE, G6PDH, HXK, and LDH, as well as levels of NADPH, ATP and hypoxanthine in sickle cells. On this basis, we conclude that impaired antioxidant capacity leaves to a loss of glycolysis and PPP shifting mechanism control and further homeostasis rupture, contributing to a decreased lifespan of sickle cells.

The impact of gas mixtures of Argon and Nitrous oxide (N2O) on quality parameters of sardine (Sardina pilchardus) fillets during refrigerated storage.

The effect of modified atmosphere packaging (MAP) with unconventional gas mixtures on the main qualitative parameters of sardine fillets during refrigerated storage was investigated. Four different atmospheres conditions were tested: air; 30% CO2 + 70% N2; 30% CO2 + 70% N2O and 30% CO2 + 70% Ar. All samples were packaged in polypropylene trays sealed with a high barrier film and stored at 2-4 °C for 12 days. The quality and the freshness of sardine fillets packed in MAP were evaluated by microbiological, physical and chemical analyses after 0, 1, 2, 5, 6, 8 and 12 days of the storage period. The 2-thiobarbituric acid-reactive substances (TBARS) values for MAP samples were lower compared to air samples, reaching a final value of 1.09 mg malonaldehyde (MA)/kg and 3.39 mg MA/kg, respectively. The samples packed in Ar reached the fixed threshold for total mesophilic and psychrotrophic bacteria after 12 days of storage, resulting the best MAP condition adopted, able to increase the sardine shelf-life of 3 days with respect to the other tested conditions. Air packed samples showed significantly higher (p < 0.05) Hx content (50 mg/kg) compared to the rest of the MAP samples (20 mg/kg). At the end of the storage period, the sample packed in Ar showed a significantly lower value (p < 0.05) (around 40 mg/kg), than the other MAP conditions.

A reliable method for estimating the postmortem interval from the biochemistry of the vitreous humor, temperature and body weight.

The estimation of the time elapsed since death is of paramount importance in the field of forensic sciences and criminal investigation, owing, among other factors, to the possible legal repercussions. Over the past few years various formulae have been developed to calculate this interval using a combination of different statistical methods and the concentrations of substances found in the vitreous humor. Corrective factors, such as ambient temperature, cause of death or age, which can modify the concentration of these substances and therefore the estimation of the postmortem interval, have been incorporated into models. In this paper five simple and reliable models to estimate PMI based the on the analysis of potassium, hypoxanthine and urea in the vitreous humor are presented. Corrective factors, such as body weight, rectal temperature and ambient temperature, which can influence the estimation of this interval have been incorporated into the formulae. Finally, the R2 and the mean squared error have been calculated for each model in order to select the best of the five. A free software program which calculates the PMI from the model and parameters used is available from the authors. It provides quick and reliable results as well as the error committed and R2 for each case.