R. Anthony Shaw

Quantitation of Protein, Creatinine, and Urea in Urine by Near-Infrared Spectroscopy

OBJECTIVES To determine the feasibility of near-infrared analysis for quantitating urea, creatinine, and protein in urine. Practical advantages of this method include ease of sample presentation and the absence of reagents or disposables. DESIGN AND METHODS The near-infrared methods were developed by first measuring the spectra of 123 different urine samples and, using independent clinical analyses, determining the protein, creatinine, and urea levels in each. Calibration models relating near-infrared spectroscopic features to those independently determined concentrations were optimized, and each model then validated using a set of 50 additional samples. RESULTS Standard errors of calibration were 14.4 mmol/L, 0.66 mmol/L, and 0.20 g/L, and standard errors of prediction 16.6 mmol/L, 0.79 mmol/L, and 0.23 g/L, respectively, for urea, creatinine, and protein. CONCLUSIONS Near-infrared urea quantitation is as accurate as the reference method, enzymatic (urease) conductivity, used here for calibration. Creatinine analysis is slightly less accurate relative to the reference (Jaffe rate) method; however, these errors can be minimized by careful attention to factors affecting precision. The accuracy of the near-infrared protein analysis cannot approach that of the reference method; nevertheless, the technique is potentially useful for coarse screening and for quantifying protein levels above 0.3 g/L.

Vibrational spectroscopy and medicine: an alliance in the making

Both infrared (IR) and Raman spectroscopy are emerging as powerful probes of biomedically relevant properties of tissue and biological fluids. From tentative first steps, this field of endeavor is now beginning to mature as the central conceptual and technical issues come into focus. Using representative examples mainly from our own research, the aim of the present article is to provide the reader with a brief overview of progress to date.

Vibrational biospectroscopy: from plants to animals to humans. A historical perspective

Abstract Today, more than ever, vibrational spectroscopy means different things to different people. From their roots as molecular fingerprinting techniques, both infrared and Raman spectroscopy have evolved to the point where they play roles in a staggering variety of scientific endeavors. This survey focuses upon biological and medical applications. The past 40 years have witnessed enormous advances in our understanding of the building blocks of life, and vibrational spectroscopy has played an important role. That role is reviewed briefly here. In parallel with these efforts, the near-IR community developed powerful ‘chemometric’ methods to extract a wealth of information from spectra that appeared superficially featureless. As vibrational spectroscopy is finding new niches in the medical and clinical realms, these chemometric methods are proving to be a valuable (but not infallible!) adjunct to conventional spectral interpretation. This survey includes a brief outline of biomedical vibrational spectroscopy and imaging, including several representative examples to illustrate the strengths and pitfalls of a growing reliance upon multivariate quantitation and classification methods.

Relative Contributions of Hemoglobin and Myoglobin to Near-Infrared Spectroscopic Images of Cardiac Tissue:

Near-infrared (NIR) spectroscopic imaging is emerging as a unique tool for intra-operative assessment of myocardial oxygenation, but quantitative interpretation of the images is not straightforward. One confounding factor specific to muscle tissue (both skeletal and cardiac) is that the visible/NIR absorbance spectrum of myoglobin (Mb), an intracellular O2 storage protein, is virtually identical to that of hemoglobin (Hb). As a consequence, the relative contributions of Mb and Hb to the NIR spectra measured in vivo for blood perfused muscle tissue cannot be determined from the measured spectra alone. To estimate the relative contributions of Mb and Hb to NIR spectra and spectroscopic images, isolated pig hearts were perfused first with a Hb-free blood substitute (Krebs-Henseleit buffer; KHB) and then with a 50/50 KHB/blood mixture, with spectroscopic images acquired at each step. Tissue Mb levels were estimated directly from the measurements during KHB perfusion, and total (Mb+Hb) levels were estimated from the images acquired during 50/50 blood/KHB perfusion. Myoglobin accounted for 63 ± 11% of the total heme content during perfusion with the 50/50 mixture (implying that Mb would contribute 46% of the combined (Mb+Hb) NIR profile during whole blood perfusion), confirming that Mb contributes substantially to near-infrared absorbance spectra of blood perfused cardiac tissue.

Infrared Spectroscopic Identification of β-Thalassemia

Background: The aim of this study was to investigate the potential of infrared (IR) spectroscopy as a fast and reagent-free adjunct tool in the diagnosis and screening of β-thalassemia. Methods: Blood was obtained from 56 patients with β-thalassemia major, 1 patient with hemoglobin H disease, and 35 age-matched controls. Hemolysates of blood samples were centrifuged to remove stroma. IR absorption spectra were recorded for duplicate films dried from 5 μL of hemolysate. Differentiation between the two groups of hemoglobin spectra was by two statistical methods: an unsupervised cluster analysis and a supervised linear discriminant analysis (LDA). Results: The IR spectra revealed changes in the secondary structure of hemoglobin from β-thalassemia patients compared with that from controls, in particular, a decreased α-helix content, an increased content of parallel and antiparallel β-sheets, and changes in the tyrosine ring absorption band. The hemoglobin from β-thalassemia patients also showed an increase in the intensity of the IR bands from the cysteine −SH groups. The unsupervised cluster analysis, statistically separating spectra into different groups according to subtle IR spectral differences, allowed separation of control hemoglobin from β-thalassemia hemoglobin spectra, based mainly on differences in protein secondary structure. The supervised LDA method provided 100% classification accuracy for the training set and 98% accuracy for the validation set in partitioning control and β-thalassemia samples. Conclusion: IR spectroscopy holds promise in the clinical diagnosis and screening of β-thalassemia.

Multianalyte Serum Assays from Mid-IR Spectra of Dry Films on Glass Slides

An analytical method based upon mid-infrared spectroscopy is proposed, and the advantages of this approach are discussed. The method involves drying a liquid specimen to a film, and deriving analyte levels from the infrared spectrum of that film. The specific aim of this study was to determine whether glass might serve as a suitable substrate for the simultaneous determination of several analytes in complex mixtures. Using human serum as a “proof-of-concept” example, we show here that six commonly measured analytes may be determined from spectra originally measured by employing barium fluoride substrates, but restricting the analytical models to absorptions within the region 2000–4000 cm−1—i.e., making use of only those absorptions that are accessible with glass substrates. With the use of partial least-squares calibration models, it is shown that albumin, cholesterol, glucose, total protein, triglycerides, and urea may be determined with standard errors that approach or meet the criteria required for routine clinical analysis. The practical advantages of such an approach are discussed.

Regional cardiac tissue oxygenation as a function of blood flow and pO2: a near-infrared spectroscopic imaging study

Near-infrared spectroscopic imaging (NIRSI) is useful to assess cardiac tissue oxygenation in arrested and beating hearts, and it shows potential as an intraoperative gauge of the effectiveness of bypass grafting. The purpose of this study was to determine whether NIRSI can reliably differentiate among a range of cardiac oxygenation states, using ischemia and hypoxia models independently. An ischemia-reperfusion model was applied to isolated, beating, blood-perfused porcine hearts, in which the left anterior descending (LAD) artery was cannulated. LAD flow was decreased stepwise to approximately 50, 20, and 0% of normal flow and was completely restored between ischemic episodes. Upon completion of the ischemia-reperfusion protocol, the hearts were further subjected to periods of increasingly severe global hypoxia. Regional oxy- and deoxy-hemoglobin (myoglobin) levels were derived from spectroscopic images (650 to 1050 nm) acquired at each step. Oxygenation maps vividly highlighted the area at risk for all degrees of ischemia. Oxygenation values differed significantly for different LAD flow rates, regardless of whether intermediate reperfusion was applied, and oxygenation values during progressive hypoxia correlated well with blood oxygen saturation. These results suggest that NIRSI is well suited, not only to identify ischemic or hypoxic regions of cardiac tissue, but also to assess the severity of deoxygenation.

Assessment of optical path length in tissue using neodymium and water absorptions for application to near-infrared spectroscopy

Quantitative analysis of blood oxygen saturation using near-IR spectroscopy is made difficult by uncertainties in both the absolute value and the wavelength dependence of the optical path length. We introduce a novel means of assessing the wavelength dependence of path length, exploiting the relative intensities of several absorptions exhibited by an exogenous contrast agent (neodymium). Combined with a previously described method that exploits endogenous water absorptions, the described technique estimates the absolute path length at several wavelengths throughout the visible/near-IR range of interest. Isolated rat hearts (n = 11) are perfused separately with Krebs-Henseleit buffer (KHB) and a KHB solution to which neodymium had been added, and visible/near-IR spectra are acquired using an optical probe made up of emission and collection fibers in concentric rings of diameters 1 and 3 mm, respectively. Relative optical path lengths at 520, 580, 679, 740, 800, 870, and 975 nm are 0.41+/-0.13, 0.49+/-0.21, 0.90+/-0.09, 0.94+/-0.01, 1.00, 0.84+/-0.01, and 0.78+/-0.08, respectively. The absolute path length at 975 nm is estimated to be 3.8+/-0.6 mm, based on the intensity of the water absorptions and the known tissue water concentration. These results are strictly valid only for the experimental geometry applied here.

Exploration of attenuated total reflectance mid-infrared spectroscopy and multivariate calibration to measure immunoglobulin G in human sera

Immunoglobulin G (IgG) is crucial for the protection of the host from invasive pathogens. Due to its importance for human health, tools that enable the monitoring of IgG levels are highly desired. Consequently there is a need for methods to determine the IgG concentration that are simple, rapid, and inexpensive. This work explored the potential of attenuated total reflectance (ATR) infrared spectroscopy as a method to determine IgG concentrations in human serum samples. Venous blood samples were collected from adults and children, and from the umbilical cord of newborns. The serum was harvested and tested using ATR infrared spectroscopy. Partial least squares (PLS) regression provided the basis to develop the new analytical methods. Three PLS calibrations were determined: one for the combined set of the venous and umbilical cord serum samples, the second for only the umbilical cord samples, and the third for only the venous samples. The number of PLS factors was chosen by critical evaluation of Monte Carlo-based cross validation results. The predictive performance for each PLS calibration was evaluated using the Pearson correlation coefficient, scatter plot and Bland-Altman plot, and percent deviations for independent prediction sets. The repeatability was evaluated by standard deviation and relative standard deviation. The results showed that ATR infrared spectroscopy is potentially a simple, quick, and inexpensive method to measure IgG concentrations in human serum samples. The results also showed that it is possible to build a united calibration curve for the umbilical cord and the venous samples.

Measurement of serum immunoglobulin G in dairy cattle using Fourier-transform infrared spectroscopy: A reagent free approach

Simple, rapid and cost-effective methods are sought for measuring immunoglobulin G (IgG) concentrations in bovine serum, which can be applied for diagnosis of failure of transfer of passive immunity (FTPI). The aim of the present study was to investigate the potential use of Fourier-transform infrared (FTIR) spectroscopy, with partial least squares (PLS) regression, to measure IgG concentrations in bovine serum. Serum samples collected from calves and adult cows were tested in parallel by radial immunodiffusion (RID) assay and FTIR spectroscopy. The sample IgG concentrations obtained by the RID method were linked to pre-processed spectra and divided into two sets: a combined set and a test set. The combined set was used for building a calibration model, while the test set was used to assess the predictive ability of the calibration model, resulting in a root mean squared error of prediction (RMSEP) of 307.5 mg/dL. The concordance correlations between the IgG measured by RID and predicted by FTIR spectroscopy were 0.96 and 0.93 for the combined and test data sets, respectively. Analysis of the data using the Bland-Altman method did not show any evidence of systematic bias between FTIR spectroscopy and RID methods for measurement of IgG. The clinical applicability of FTIR spectroscopy for diagnosis of FTPI was evaluated using the entire data set and showed a sensitivity of 0.91 and specificity of 0.96, using RID as the reference standard. The FTIR spectroscopy method, described in the present study, demonstrates potential as a rapid and reagent-free tool for quantification of IgG in bovine serum, as an aid to diagnosis of FTPI in calves.