Multicomponent analysis of near-infrared spectra of anesthetized rat head: (I). Estimation of component spectra by principal component analysis.

By measuring precise NIR spectra of anesthetized rat head, we determined principal components included in changes of the NIR spectra caused by acute hypoxia. There was apparent delay in reduction of cyt. aa3 as compared with Hb deoxygenation in the hypoxic period. With regard to the eigenvalues determined by principal component analysis, principal components in the NIR spectral changes caused by acute hypoxia were Hb and cyt. aa3, and contribution of the remaining biological materials to the changes in NIR spectra caused by acute hypoxia was considered to be negligible in quantitative multivariate analysis of Hb and cyt. aa3 in situ.

31P nuclear magnetic resonance spectroscopy studies of tumor energy metabolism and its relationship to intracapillary oxyhemoglobin saturation status and tumor hypoxia.

Relationships between tumor bioenergetic status on the one hand and intracapillary oxyhemoglobin (HbO2) saturation status and fraction of radiobiologically hypoxic cells on the other were studied using two murine sarcoma lines (KHT, RIF-1) and two human ovarian carcinoma xenograft lines (MLS, OWI). Tumor energy metabolism was studied in vivo by 31P nuclear magnetic resonance (NMR) spectroscopy and the resonance area ratio (PCr + NTP beta)/Pi was used as parameter for bioenergetic status. Intracapillary HbO2 saturation status reflects the oxygen supply conditions in tumors and was measured in vitro using a cryospectrophotometric method. The KHT, RIF-1, and MLS lines showed decreasing bioenergetic status, i.e., decreasing PCr and NTP beta resonances and an increasing Pi resonance, with increasing tumor volume, whereas the OWI line showed no changes in these resonances during tumor growth. The volume-dependence of the HbO2 saturation status differed similarly among the tumor lines; HbO2 saturation status decreased with increasing tumor volume for the KHT, RIF-1, and MLS lines and was independent of tumor volume for the OWI line. Moreover, linear correlations were found between bioenergetic status and HbO2 saturation status for individual tumors of the KHT, RIF-1, and MLS lines. These observations together indicated a direct relationship between 31P-NMR spectral parameters and tumor oxygen supply conditions. However, this relationship was not identical for the different tumor lines, suggesting that it was influenced by intrinsic properties of the tumor cells such as rate of respiration and ability to survive under hypoxia. Similarly, there was no correlation between bioenergetic status and fraction of radiobiologically hypoxic cells across the four tumor lines. This indicates that 31P-NMR spectroscopy data have to be supplemented with other data, e.g., rate of oxygen consumption, cell survival time under hypoxic stress, and/or fraction of metabolically active, nonclonogenic hypoxic cells, to be useful in quantitative determination of tumor hypoxia and hence prediction of tumor radioresistance caused by hypoxia.

Direct spectrophotometry of bilirubin in serum of the newborn, with use of caffeine reagent.

We recently described a direct spectrophotometric method for unconjugated bilirubin, with caffeine reagent (Clin Chem 1986;32:1389-93). Because this method is independent of the protein matrix we used it for the preparation of bilirubin standards (Clin Chem 1987;33:1817-21). Now, in this paper, we utilize the caffeine reagent in setting up a bilirubin method for serum from neonates. This resulted in a two-wavelength (465 and 528 nm) equation, which fully corrects for HbO2 interferences. In combination with a bilirubin standard, this equation may be transformed into a simple relative formula for use with this simple dilution method. We studied this two-wavelength method with 55 neonates' sera, comparing results with those by both the diazo method of Doumas et al. (Clin Chem 1985;31:1779-89) and the borate method of Hertz et al. (Scand J Clin Lab Invest 1974;33:215-30). We found that this new method is independent of hemolysis and of the matrix of the sera. Therefore, it is very suitable for use in neonatology.

The influence of nitrous oxide on oxyhaemoglobin dissociation and measurement of oxygen tension.

There are conflicting reports of the effect of nitrous oxide on the oxyhaemoglobin dissociation curve. We have therefore determined P50 of haemoglobin in the presence of either nitrous oxide or nitrogen and studied the upper portion of the curve in greater detail. No significant differences in the oxyhaemoglobin dissociation curve were observed when nitrous oxide was substituted for nitrogen. The oxygen tensions measured in gas mixtures were not significantly different when determined simultaneously with a polarographic oxygen electrode and the mass spectrometer when nitrous oxide was used instead of nitrogen.

Temperature effects on oxygen affinity of human fetal blood.

In an effort to understand the effects of temperature changes on fetal oxygenation, the temperature effects were measured on oxygen affinity of whole blood from term human fetuses. The blood obtained was tonometered at delivery in two flasks gassed with 95% N2 (+ 5% CO2 or 20.9% + 5% CO2, and mixed aliquots from each flask in different proportions to obtain samples for analysis of PO2 and percent saturation. The oxyhaemoglobin dissociation curve was constructed and P50 determined at two or three different temperatures for each batch of blood. As temperature increased from 30 to 41 degrees C, human fetal blood bound O2 less avidly, the temperature coefficient for changes in P50 being 0.0255 per degree C. This temperature effect was similar to that in adult blood, although at any temperature O2 affinity of fetal blood was greater than that of the adult. Placental oxygen exchange could be significantly affected by changes in temperature such as occur during hypo- or hyperthermia, as with maternal exercise.

A microcomputer system for monitoring and analysing oxyhemoglobin saturation during sleep.

A computerized data acquisition and analysis routine was developed to quantitate respiratory disturbances in sleeping patients. Polysomnographic recordings of patients consisted of electroencephalograms, electro-occulograms, submental electromyograms, air flow at the nose and mouth, esophageal pressure, and oxyhemoglobin saturation (SaO2). SaO2, a physiological effect of ventilatory airflow, was sampled every two seconds and stored on disk during the night's study for subsequent analysis. Wild points in the data file can be marked so that they will be skipped during analysis. Patient polysomnographs were scored manually for sleep stage by a sleep technician. A file was then created containing the scored sleep information with time marks corresponding to each change in sleep stage during the study. An analysis routine used this file to develop indices of sleep apnea, severity for combinations or specific stages of sleep. These indices were: (A) number of oxyhemoglobin desaturation episodes per hour; (B) average maximum desaturation per episode; and (C) desaturation index, the product of (A) and (B). A graph was plotted showing cumulative sleep time at given SaO2 values. The degree of sleep apnea can be determined using these indices.

Incorporation of inositol hexaphosphate into red blood cells mediated by dimethyl sulfoxide.

Inositol hexaphosphate (IHP) binds to deoxyhemoglobin and markedly decreases the affinity of hemoglobin for oxygen. We introduce here a method for incorporating this polyphosphate into erythrocytes, thus preparing very low affinity cells for use in respiration research. The method uses dimethyl sulfoxide (DMSO) to facilitate entry of IHP. The cells are exposed to a high concentration of DMSO which is rapidly diluted with IHP solution. During this dilution the cells become leaky and IHP enters. The influence of several variables at each step of the process has been investigated and the data support a transient osmotic gradient mechanism for IHP incorporation.