James J. Miller

Brain lactate is an obligatory aerobic energy substrate for functional recovery after hypoxia : Further in vitro validation

Abstract: This study used the rat hippocampal slice preparation and the monocarboxylate transporter inhibitor, α‐cyano‐4‐hydroxycinnamate (4‐CIN), to assess the obligatory role that lactate plays in fueling the recovery of synaptic function after hypoxia upon reoxygenation. At a concentration of 500 µM, 4‐CIN blocked lactate‐supported synaptic function in hippocampal slices under normoxic conditions in 15 min. The inhibitor had no effect on glucose‐supported synaptic function. Of control hippocampal slices exposed to 10‐min hypoxia, 77.8 ± 6.8% recovered synaptic function after 30‐min reoxygenation. Of slices supplemented with 500 µM 4‐CIN, only 15 ± 10.9% recovered synaptic function despite the large amount of lactate formed during the hypoxic period and the abundance of glucose present before, during, and after hypoxia. These results indicate that 4‐CIN, when present during hypoxia and reoxygenation, blocks lactate transport from astrocytes, where the bulk of anaerobic lactate is formed, to neurons, where lactate is being utilized aerobically to support recovery of function after hypoxia. These results unequivocally validate that brain lactate is an obligatory aerobic energy substrate for posthypoxia recovery of function.

Brain lactate, not glucose, fuels the recovery of synaptic function from hypoxia upon reoxygenation: an in vitro study.

Lactate has been considered for many years to be a useless, and frequently, harmful end-product of anaerobic glycolysis. In the present in vitro study, lactate-supplied rat hippocampal slices showed a significantly higher degree of recovery of synaptic function after a short hypoxic period than slices supplied with an equicaloric amount of glucose. More importantly, all slices in which anaerobic lactate production was enhanced by pre-hypoxia glucose overload exhibited functional recovery after a prolonged hypoxia. An 80% recovery of synaptic function was observed even when glucose utilization was blocked with 2-deoxy-D-glucose during the later part of the hypoxic period and during reoxygenation. In contrast, slices in which anaerobic lactate production was blocked during the initial stages of hypoxia did not recover their synaptic function upon reoxygenation despite the abundance of glucose and the removal of 2-deoxy-D-glucose. Thus, for brain tissue to show functional recovery after prolonged period of hypoxia, the aerobic utilization of lactate as an energy substrate is mandatory.

Towards a better understanding of heterophile (and the like) antibody interference with modern immunoassays.

BACKGROUND Heterophile antibodies interfere with immunoassays. Understanding the nature and characteristics of these antibodies provides a format for better identifying and removing them. Growing evidence suggests many of these antibodies are natural antibodies. Very large number of tests are being performed with automated analyzers and there has been a problem with misdiagnosis due to interference. New commercial agents for blocking heterophile antibodies have been developed. METHODS Review of the immunology and methodological literature with critical interpretation of the findings. CONCLUSIONS Heterophile antibodies consist of natural antibodies and autoantibodies. Both types are usually weak antibodies that interfere by noncompetitive mechanisms. Based on very strong circumstantial evidence, we propose that natural antibodies account for most interference with automated immunoassays. In terms of false positive results, the interference rate is very low, about 99.95% accuracy. Specific blocking agents have some theoretical advantage over nonspecific blocking agents, but in actual practice, the very low false positive frequency makes it difficult if not impossible to statistically compare blocking agents or other assay modifications with adequate statistical power. In the absence of a technique that lends itself to automation for removing all immunoglobulins, it appears that infrequent heterophile interference cannot be avoided.

Glia are the main source of lactate utilized by neurons for recovery of function posthypoxia.

Experiments are described in which a rat hippocampal slice preparation was used along with the metabolic glial inhibitor, fluorocitrate (FC), to investigate the role of glial-made lactate and its shuttling to neurons in posthypoxia recovery of synaptic function. After testing two less effective concentrations of FC, only 10.1 +/- 6.5% of slices treated with 100 microM of the metabolic toxin recovered synaptic function at the end of 10-min hypoxia and 30-min reoxygenation. In contrast, 79.6 +/- 7.4% of control, untreated slices recovered synaptic function after 10-min hypoxia and 30-min reoxygenation. The low rate of recovery of synaptic function posthypoxia in FC-treated slices occurred despite the abundance of glucose present in the medium before, during, and after hypoxia. The amount of lactate produced by FC-treated slices during the hypoxic period was only 62% of that produced by control, untreated slices. Supplementing FC-treated slices with exogenous lactate significantly increased the posthypoxia recovery rate of synaptic function. These results strongly support our previous findings concerning the mandatory role of lactate as an aerobic energy substrate for the recovery of synaptic function posthypoxia and clearly show that the bulk of the lactate needed for this recovery originates in glial cells.

Blockade of lactate transport exacerbates delayed neuronal damage in a rat model of cerebral ischemia.

Studies over the past decade have demonstrated that lactate is produced aerobically during brain activation and it has been suggested to be an obligatory aerobic energy substrate postischemia. It has been also hypothesized, based on in vitro studies, that lactate, produced by glia in large amounts during activation and/or ischemia/hypoxia, is transported via specific glial and neuronal monocarboxylate transporters into neurons for aerobic utilization. To test the role of lactate as an aerobic energy substrate postischemia in vivo, we employed the cardiac-arrest-induced transient global cerebral ischemia (TGI) rat model and the monocarboxylate transporter inhibitor alpha-cyano-4-hydroxycinnamate (4-CIN). Once 4-CIN was establish to cross the blood--brain barrier, rats were treated with the inhibitor 60 min prior to a 5-min TGI. These rats exhibited a significantly greater degree of delayed neuronal damage in the hippocampus than control, untreated rats, as measured 7 days post-TGI. We concluded that intra-ischemically-accumulated lactate is utilized aerobically as the main energy substrate immediately postischemia. Blockade of lactate transport into neurons prevents its utilization and, consequently, exacerbates delayed ischemic neuronal damage.

Influence of obesity on vitamin D–binding protein and 25-hydroxy vitamin D levels in African American and white women

25-Hydroxy vitamin D (25OHD) is lipophilic and highly bound to vitamin D-binding protein (VDBP) in plasma. In the present study, we examined VDBP and 25OHD levels by race and body mass index (BMI) in young adult women to determine whether circulating VDBP plays a role in the low levels of 25OHD with obesity and among African Americans. In agreement with previous studies, mean 25OHD levels were lower in African American women than in whites (P < .01). In a hierarchical multiple regression model, BMI was associated with 25OHD after adjustment for age in white women (P = .02, R(2) = .10) but not in African American women. The VDBP levels, by contrast, were similar in African Americans and whites, and were unrelated to BMI in either racial group. Furthermore, VDBP was unrelated to the plasma level of 25OHD. These data confirm an interaction between race and obesity in vitamin D metabolism, and imply that the carrier protein is not an important determinant of circulating 25OHD in women, nor is it affected by race or adiposity.

Correlations among inflammatory markers in plasma, saliva and oral mucosal transudate in post-menopausal women with past intimate partner violence.

The relationship between psychosocial factors and an increased risk for disease has been related to a heightened pro-inflammatory status reflected in increased circulating levels of pro-inflammatory cytokines and/or C-reactive protein (CRP). Routinely, epidemiological studies rely on measurements of inflammatory markers in serum or plasma, but the use of biological fluids such as saliva or oral mucosal transudate (OMT) may offer potential advantages. This study investigated correlations among plasma CRP and levels of IL-6 and soluble IL-6 receptor (sIL-6R) in plasma, saliva and OMT in a population of middle aged women with histories of past intimate partner violence (IPV). A total of 67 women without existing chronic diseases participated in the study, which included two visits each in which psychological tests were administered, and blood, saliva and OMT samples were collected. Although significantly higher plasma CRP levels were found in past IPV sufferers compared to controls, there were no significant differences in IL-6 or sIL-6R levels in plasma, saliva or OMT between the two groups. There were only relatively modest correlations between IL-6 levels in plasma and those in saliva or OMT and between plasma IL-6 and CRP levels. A significant correlation between IL-6 and sIL-6R levels in both saliva and OMT, but not in plasma, was also detected. No significant correlations were found between levels of IL-6 in saliva or OMT and periodontal health measures. Results indicate that IL-6 and sIL-6R levels in saliva or OMT do not closely reflect those in plasma, and therefore are not a good surrogate for systemic levels.

Preischemic hyperglycemia-aggravated damage: Evidence that lactate utilization is beneficial and glucose-induced corticosterone release is detrimental

Aerobic lactate utilization is crucial for recovery of neuronal function posthypoxia in vitro. In vivo models of cerebral ischemia pose a conceptual challenge when compared to in vitro models. First, the glucose paradox of cerebral ischemia, namely, the aggravation of delayed neuronal damage by preischemic hyperglycemia, cannot be reproduced in vitro. Second, in vitro elevated glucose levels protect against ischemic (hypoxic) damage, an outcome that has seldom been reproduced in vivo. Employing a rat model of cardiac‐arrest‐induced transient global cerebral ischemia (TGI), we found that hyperglycemic conditions, when induced 120–240 min pre‐TGI, significantly reduced post‐TGI neuronal damage as compared to normoglycemic conditions. In contrast, hyperglycemia, when induced 15–60 min pre‐TGI, significantly aggravated post‐TGI neuronal damage. Brain lactate levels in rats loaded with glucose either 15 min or 120 min pre‐TGI were significantly and equally higher than those of control, saline‐injected rats. The beneficial effect of 120 min pre‐TGI glucose loading was abolished by lactate transport inhibition. A significant increase in blood corticosterone (CT) levels was observed upon glucose loading that peaked at 15–30 min and returned to baseline levels by 60–120 min. When rats loaded with glucose 15 min pre‐TGI were treated with metyrapone, a CT synthesis inhibitor, a significantly lower degree of delayed neuronal damage in comparison to both untreated, 15 min glucose‐loaded rats and normoglycemic, control rats was observed. Thus, although elevated levels of brain lactate cannot explain the glucose paradox of cerebral ischemia, hyperglycemia‐induced, short‐lived elevation in CT blood levels could. More importantly, lactate appears to play a crucial role in improving postischemic outcome.

Endogenous digoxin-like immunoreactive factor (DLIF) serum concentrations are decreased in manic bipolar patients compared to normal controls

BACKGROUND A decrease in sodium pump activity in erythrocytes has been associated with manic episodes of bipolar illness relative to euthymic moods. Since red blood cells are long-lived and lack a nucleus, it is likely that a plasma factor is responsible for the observed decrease in sodium pump activity. METHODS Utilizing a radioimmunoassay, we examined the serum concentrations of the digoxin-like immunoreactive factor (DLIF) in ill and well bipolar patients and compared the values to those of normal controls. RESULTS DLIF was significantly decreased in manic individuals as compared to normal controls (143.6+/-S.E.M. 20.94 vs. 296.6+/-12.76 pg digoxin equivalents/ml, respectively, F = 4.77, P<0.05), but not compared to euthymic bipolar subjects 213.8+/-86.92, P = 0.77). There were no significant differences in DLIF concentrations between manic and euthymic bipolar individuals (P = 0.8). Since relapse in bipolar patients appears to display a seasonal pattern, we also measured the plasma concentration of this factor over a 12-months period. Normal controls exhibited a seasonal pattern of change in serum DLIF concentrations with a nadir in the winter months. Plasma concentrations of DLIF in bipolar patients did not show a seasonal pattern and maintained low levels throughout the year. LIMITATIONS Due to the nonspecificity of our antibody, we could measure only total DLIF. Furthermore, it is unclear what the role of circulating DLIF, if any, may be on brain function. CONCLUSION DLIF may be involved in the pathophysiology of mania.

Audiogenic seizures following global ischemia induced by chest compression in Long-Evans rats

Transient global ischemia was used to produce a rat model of generalized tonic-clonic epilepsy. Controlled chest compression in ketamine-anesthesized Long-Evans rats produced transient global ischemia by mechanically preventing the heart from pumping blood. Circulation was restored by standard cardiopulmonary resuscitation techniques. With a temporal muscle (skull) temperature of 35 +/- 0.4 degrees C, 75% (76/102) of the rats survived 7 min of chest compression. Generalized seizures could be evoked in 78% (59/76) of the surviving rats by a 60 s exposure to a loud sound (bell, 110 dB) beginning 24 h after the ischemic episode. The seizure patterns seen resembled those described by Maresceaux (1987) for genetically seizure-prone Wistar rats. Susceptibility to sound-induced seizures declined with time, with wide variations in recovery rate between individuals; one rat showed a daily sound-induced seizure for over 5 months. Seizures were attenuated or blocked by treatment with carbamazepine or sodium valproate. This model is similar to the great vessel occlusion model used by Kawai et al. (1995), but is less invasive. We believe it will be useful in the evaluation of therapies for acquired generalized (grand mal) seizures.