The Acid-Base Status of the Blood

Circadian rhythms organize many aspects of cell biology and physiology to a daily temporal program that depends on clock gene expression cycles in most mammalian cell types. However, circadian rhythms are also observed in isolated mammalian red blood cells (RBCs), which lack nuclei, suggesting the existence of post-translational cellular clock mechanisms in these cells. Here we show using electrophysiological and pharmacological approaches that human RBCs display circadian regulation of membrane conductance and cytoplasmic conductivity that depends on the cycling of cytoplasmic K+ levels. Using pharmacological intervention and ion replacement, we show that inhibition of K+ transport abolishes RBC electrophysiological rhythms. Our results suggest that in the absence of conventional transcription cycles, RBCs maintain a circadian rhythm in membrane electrophysiology through dynamic regulation of K+ transport.

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Rhythmic potassium transport regulates the circadian clock in human red blood cells

UNLABELLED The aim of this paper is a critical reflection of the practice in competitive cycling to use the hematocrit value (Hct) as an indirect control measure for doping with erythropoietin. To demonstrate the individual physiological variation of Hct values, five different studies were performed: 1) Eight subjects were observed (i) during 23 h after a 1 h lasting bout of cycle exercise at 60% of maximum performance and (ii) during 24h under control conditions. 2) Seven subjects were exposed to a 20 min period of -7 head down tilt (HDT), which was followed by 15 min in sitting position. 3) From four subjects blood samples were taken in a sitting position up to 60 min after they had ingested 1 liter isotonic saline solution. 4) Ten subjects performed a vita maxima test on a cycle ergometer, starting at 100W and increasing the workload by 17W every minute. 5) Four elite cyclists participated in a 10 days competition (1,700 km). RESULTS 1) During the 24h observation period Hct decreased during the night from 45.3+/-3.1 % to 42.9+/-1.5% and returned to the initial values in the morning. This diurnal variation was even more pronounced after submaximal exercise (-4.1 %). 2) Due to fluid shifts from the interstitial into the intravasal compartment, HDT was accompanied by a 3.1+/-0.5% lower Hct. 3) Drinking of the isotonic saline solution also reduced the hematocrit by 3.3+/-0.5% after one hour. 4) Maximum cycle exercise increased the Hct from 46.8+/-2.4 % to 51.3+/-1.9% which was due to a 15 % decrease in plasma volume. 5) Repeated bouts of cycle-exercise reduced the Hct from 46.4+/-1.5% to 41.3+/-1.6%. CONCLUSIONS All experiments demonstrate that the Hct is not a constant value but can be considerably changed by physiological measures. Clinical studies show that brain oxygen supply decreases with increasing Hct-values, which are also associated with a higher risk of stroke accidents. We therefore recommend to use a Hct-limit solely under strongly controlled standardized conditions to protect professional cyclists from hazardous manoeuvre until more appropriate methods to detect EPO-doping are developed.

How Valid is the Determination of Hematocrit Values to Detect Blood Manipulations

1 Introduction.- 2 Two Gas Transport Problems.- Requirements at Rest.- Oxygen.- Carbon Dioxide.- Equilibrium Binding Curves.- Requirements during Exercise.- Some Further Requirements.- Regulation.- Darwinian Evolution in the Genealogy of Hemoglobin.- Why Red Cells?.- 3 Physical Relationships in Blood-Tissue Gas Transport.- 4 Blood as a Physicochemical System-I. Hemoglobin and Its Interaction with Ligands.- Hemoglobin Structure.- Ligand Binding and Conformational States.- The Monod-Wyman-Changeux Model.- Adair's Intermediate Compound Hypothesis.- Linked Functions.- The Molecular Basis of Ligand Binding, Conformational Change, and Allosteric Interaction of the Hemoglobin Molecule.- Oxygen.- Protons.- Carbon Dioxide.- 2,3-Diphosphoglycerate.- Carbon Monoxide.- Oxygen Binding and Interaction Coefficients under Physiological Conditions.- Basic Considerations.- The Whole Blood Oxygen Binding Curve under Standard Conditions.- Numerical Values.- Effect of Hemoglobin Concentration.- Carbon Monoxide.- Effect of Temperature.- Embryonic and Fetal Hemoglobins.- Hemoglobin-Ligand Kinetics in Solution.- Hemoglobin-Oxygen Interaction.- Hemoglobin Interactions with CO2, Protons, and 2,3- DPG.- 5 Blood as a Physicochemical System-II. Equilibria, Steady States, and Transport Kinetics of the Red Cell-Plasma Distributions.- Definition of a Standard State for Measurements of Blood Composition.- The Standard Steady State Distributions.- Red Cell Water.- Red Cell Ionic Composition.- Thermodynamic Equilibria and Ion Activity Coefficients.- Erythrocyte and Plasma Responses to Perturbations in Composition.- Fundamental Constraints and Approach to Analysis.- Applications of the Fundamental Relationships.- Review of Data on the Kinetics of Relaxation Processes in Blood.- 6 The Dynamics of Oxygen, Carbon Dioxide, and Proton Transport under Normal Conditions.- The Model System.- Geometric Considerations.- Analysis of Plasma and Red Cell Compositional Profiles.- Determination of Systemic Capillary Blood Flowrate.- Simulation of Gas Transport at Rest.- Base Case.- A Simplifying Approximation.- Oxygen Transport in Exercise.- Facilitated Gas Transport in Tissue.- Oxygen-Myoglobin.- Carbon Dioxide-Bicarbonate.- 7 Some Physiological Control Systems.- The Controlled Gas Transport System.- The Red Cell 2,3-DPG Concentration.- 2,3-DPG and the Red Cell Glycolytic System.- Variations in Red Cell 2,3-DPG Concentration under Physiological Conditions.- The Whole Blood Hemoglobin Concentration.- General Aspects.- The Erythropoietin System.- Variations under Physiological Conditions.- Protection against Oxidation of Hemoglobin.- The Heme Groups.- The-SH Groups.- 8 Disturbances of the Respiratory Functions of Blood.- Abnormal Acid-Base Balance.- General Discussion and Results.- The in Vivo Dependence of Red Cell 2,3-DPG Level, [DPG]T, upon pH and Hemoglobin Oxygen Saturation.- Abnormal Erythron function.- Anemia.- Oxygen Affinity and Red Cell Age.- Kinetics of Oxygen Transfer in Red Cells with Increased Rigidity.- Polycythemia.- Abnormal Hemoglobins.- Abnormal Red Cell Organic Phosphate Concentration.- Carboxyhemoglobin and Methemoglobin.- Stored Red Cells.- Abnormal Lung Function and Ambient Air.- Simulation of the Effects of Abnormal Arterial Gas Tensions.- 9 Concluding Remarks.- References.

The respiratory functions of blood

Four women were studied at 0400 h and 1600 h to determine if their hormonal and hemodynamic responses to exercise varied with the circadian cycle. Esophageal temperature was measured during rest and exercise (60% peak
 
$$\dot V_{{\text{O}}_{\text{2}} } $$

 ; 30 min) in a warm room (T
a=35° C;
 
$$P_{{\text{H}}_{\text{2}} {\text{O}}} = 1.7$$

 =1.7 kPa). Venous blood samples were drawn during rest and exercise and hemoglobin concentration (Hb), hematocrit (Hct), plasma osmolality (P
osm), plasma protein concentration (P
p), colloid osmotic pressure (COP), plasma renin activity (PRA), cortisol, aldosterone, norepinephrine (NE) and epinephrine (E) were determined. Changes in plasma volume (PV) were estimated from changes in Hb and Hct. The relative hemoconcentration (−11.2%) was similar at 0400 h and 1600 h, but the absolute PV was smaller at 1600 h than at 0400 h (p=0.03). The responses ofP
osm,P
p and COP to exercise were unaffected by time of day. Although PRA was not different at the two times of day, PRA was 244% greater during exercise at 1600 h, but only 103% greater during exercise at 0400 h. The normal circadian rhythms in plasma aldosterone (p=0.043) and plasma cortisol (p=0.004) were observed. Plasma aldosterone was 57% greater during exercise, while plasma cortisol did not change. The change in E and NE was greater at 0400 h, but this was due to the lower resting values of the catecholamines at 0400 h. These data indicate that time of day generally did not affect the hormonal or hemodynamic responses to exercise, with the exception that PRA was markedly higher during exercise at 1600 h compared to 0400 h.

Circadian variations in plasma renin activity, catecholamines and aldosterone during exercise in women.

Venous blood was sampled in 3-h intervals from 5 men and 4 women under conditions of normal daily activity during 24 h (Series I). Hematocrit, hemoglobin concentration in blood and red cells, sodium, potassium, magnesium and chloride concentrations in plasma and red cells, protein, calcium and inorganic phosphate concentrations and standard bicarbonate in plasma, pH in blood and red cells, pCO2 in blood were measured or calculated. Most concentration curves showed biphasic patterns with night minima which were significant for hemoglobin, hematocrit and pH in blood, protein, sodium, calcium and standard bicarbonate in plasma, potassium and pH in red cells. Inorganic phosphate in plasma alone exhibited a definite day minimum. A second series (6 subjects) with measurements made only between 06.00 and 12.00 showed changes similar to those in Series I with an additional small increase of red cell sodium concentration at noon. In a third series, where 6 subjects remained in a recumbent position up to midday, the morning increase of [Hb], Hct, pH and standard bicarbonate was clearly reproducible.

Diurnal Variations of Protein and Electrolyte Concentrations and of Acid-Base Status in Plasma and Red Cells of Normal Man*

The 2,3-diphosphoglycerate (2,3-DPG) concentration, oxygen half saturation pressure at pH 7.4 (P50), pH in plasma and red cells, and mean corpuscular hemoglobin concentration (MCHC) of venous blood were determined during unrestricted daily activity (series I) throughout 24 hrs as well as during prolonged bed rest until noon (series II). In series I an almost synchronous diurnal behavior ofP50, 2,3-DPG, and plasma pH as well as red cell pH became significantly apparent with highest values in the afternoon. The [2,3-DPG] yielded most pronounced alterations, which made up to 13.5% of the average day value. During prolonged recumbency the [2,3-DPG] showed a nonsignificant tendency to decline; theP50 remained unchanged throughout that period. The possible reason for the missing [2,3-DPG] increase is a reduced change of red cell pH in series II. An influence of a posture dependent aldosterone secretion either directly on the 2,3-DPG metabolism or indirectly via mediating the red cell pH and thus ruling the formation of this organic phosphoris compound is discussed.

M. Kunze

Papers

Diurnal Variations of Protein and Electrolyte Concentrations and of Acid-Base Status in Plasma and Red Cells of Normal Man*

Diurnal changes of the 2,3-diphosphoglycerate concentration in human red cells and the influence of posture.