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Slide 1: Title Slide

Hello. My name is Dr. Dennis Dietzen. I am Associate Professor of Pediatrics at Washington University in St. Louis and Director of the Core and Metabolic Genetics Laboratories at St. Louis Children’s Hospital.

Welcome to this discussion on lactate and lactic acidemia.

Slide 2: Overview

This presentation will cover the chemistry, biochemistry, and clinical aspects of lactic acid. Generation of lactate is essential for the generation of cellular energy. Glycolysis splits glucose into two 3-carbon compounds. Electrons from the products of glycolysis are converted to energy by anaerobic fermentation or by oxidative respiration. Lactate results from fermentation while water is ultimately the result of respiration. Respiration is a far more potent energy producing process in mammalian metabolism. Accumulation of lactate is a signal of deranged metabolism. The differential diagnosis of lactic acidemia is large and accurate measurement of circulating lactate is subject to a number of pitfalls. We will discuss the causes, classification, and analytic aspects relevant to lactic acidemia.

Slide 3: Lactic Acid/Lactate Stats

Lactic acid is a 3-carbon carboxylic acid. The second or β-carbon atom (shown in red) is hydroxylated and is chiral, giving rise to 2 stereoisomers referred to as the D or L configuration. At pH 7.4, virtually all lactic acid exists as an anion and will be referred to as lactate during the remainder of this presentation. The vast majority of circulating lactate (normally present at millimolar concentrations) is in the L- configuration while only nanomolar quantities of the D isomer are present. Circulating lactate is analyzed most commonly in plasma by enzymatic-photometric techniques but may be assessed in whole blood or other body fluids by enzymatic-amperometric techniques, or by mass spectrometry. Tissue lactate is also sometimes assessed by magnetic resonance spectroscopy.

Slide 4: Whence comes lactate?

Lactate is derived from glucose via glycolysis. Glycolysis splits one 6-carbon glucose molecule into two 3- carbon compounds. The pathway is shown on the left of the slide. The terminal reaction of glycolysis generates pyruvate. The disposition of pyruvate is under tight control mediated by the energy state of the cell (ATP/ADP ratio) and the availability of acetyl CoA. For example, a plentiful supply of acetyl CoA inhibits pyruvate oxidation and promotes gluconeogenesis. Reduction of pyruvate by lactate dehydrogenase (LDH) in the cytoplasm yields lactate. Two moles of lactate may therefore be derived from one mole of glucose. Glycolysis does not require oxygen and generates 2 moles of ATP for every mole of glucose that enters the pathway. Glycolysis also converts NAD+ into NADH. NADH may be used to yield energy via indirect oxidation in mitochondria.

Slide 5: Why do we make lactate?

In prokaryotic organisms, the generation of lactate is necessary to allow continuing generation of energy from glucose. The glyceraldehyde-3-phosphate dehydrogenase step of glycolysis generates reducing equivalents by converting NAD+ to NADH. NAD (nicotine adenine dinucleotide) consists of a nicotinamide moiety linked to adenine via a phosphodiester bond and is shown in the upper right portion of the slide. This enzyme cofactor is energetically expensive to synthesize and maintain. Without re-oxidation of NADH, the cellular pool would become depleted and glycolysis would stop. The LDH reaction serves to reoxidize cytoplasmic NADH. In other organisms such as yeast, regeneration of NAD+ is accomplished by conversion of pyruvate to ethanol and CO2. In higher eukaryotes, NADH is reoxidized indirectly in the mitochondria via shuttle systems (e.g., the malate-aspartate shuttle), a process which generates substantial amounts of ATP.

Slide 6: What else can we do with lactate?

In addition to its role in conserving the cellular pool of NAD, lactate is also a key player in the recycling of glucose carbon. Pyruvate is a key intermediate in this process. Depending on the energy needs of the cell, lactate may be oxidized via pyruvate to generate ATP in the Krebs Cycle, or carboxylated to initiate gluconeogenesis. The process whereby lactate generated in muscles is converted back to glucose in the liver is referred to as the Cori Cycle. The cycle is named after Carl and Gerty Cori who received the Nobel Prize for delineation of this pathway in 1947. Generation of lactate and pyruvate that exceeds oxidative and gluconeogenic capacities of the liver are reflected in the circulating alanine concentration via the activity of alanine aminotransferase (ALT).

Slide 7: Lactic Acidosis (Fermentation > Oxidation)

Lactic acidosis results when lactate production exceeds disposal. The primary routes of disposal are oxidation, gluconeogenesis, and renal excretion. The liver accounts for the largest component of lactate extraction from blood. The kidney metabolizes a substantial amount of lactate and excretes a small fraction under normal circumstances. Renal excretion increases when circulating lactate reaches 6-10 mM. Oxidation and gluconeogenesis both require a functional respiratory apparatus. Thus, any pathologic process that impairs respiration/mitochondrial function will have an impact on the removal of lactate. Accumulation of lactate beyond the excretory capacity of the kidney results in lactic acidemia.

Slide 8: Classification of Lactic Acidosis

Lactic acidosis may be classified in many different ways. In the scheme presented on the current slide, the mechanisms of lactic acidosis are divided into primary and secondary causes. Primary causes are those that directly impact the integrity/function of the mitochondrial apparatus. These include mutations in the mitochondrial genome that encodes mitochondrial tRNA and 13 polypeptides.

Approximately 1500 nuclear genes provide translated products that function within the mitochondrion. Two of these genes encode critical enzymes that metabolize pyruvate: pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC). Mutations in these genes commonly cripple mitochondrial function.

Common secondary causes of lactic acidosis are those that limit tissue oxygenation. These include heart failure, pulmonary disease, and hemoglobinopathies, to name a few. Other secondary causes include toxins that impair mitochondrial function. Salicylates, for example, uncouple oxidative phosphorylation and cyanide inhibits terminal electron transfer to water by cytochrome oxidase. Finally, accumulation of D-lactate may occur in short bowel syndrome. In these circumstances, malabsorption of carbohydrate fuels fermentation by gut lactobacilli and produces a high-anion gap acidosis. Measurement of D-lactate must be targeted specifically as common laboratory techniques detect only the L-isomer.

Slide 9: Clinical Classification of Lactic Acidosis

Other classification systems for the lactic acidoses exist. The system detailed by Woods and Cohen is also commonly cited. Generally, type A conditions are those resulting from tissue hypoxia or hypoperfusion. The type B disorders include a host of primary and secondary causes of lactic acidosis. Type B1 includes specific organ failure and other systemic conditions such as inflammation. Type B2 includes mitotoxins, and type B3 includes primary defects in mitochondrial structure and function, as well as other metabolic disorders that impair mitochondrial metabolism.

Slide 10: Clinical Utility of Blood Lactate-1

There is not a single phenotype typical of lactic acidosis. Affected patients may be severely ill or nearly asymptomatic. The algorithm above is a very generalized approach to the patient with potential lactic acidosis. As will be discussed in subsequent slides, blood lactate concentrations are commonly overestimated. It is important to rule out artifactual elevation prior to further laboratory investigation. Once established, a series of common laboratory (e.g., ABG, hemoglobin, cooximetry) and physical findings (e.g., respiratory rate, heart rate, edema, fever) may point to a plausible mechanism. Broad toxicology screening and microbiological screening may serve to confirm and augment clinical findings. In general, lactic acidoses due to cardiac, pulmonary, toxic, or infectious etiologies beget quick supportive treatment and therapy targeted at the cause (e.g., antibiotics for infectious etiologies).

Metabolic causes are often obscure and require broad, lengthy metabolomic and genetic investigations to uncover. Primary mitochondrial pathology is further complicated by uneven tissue distribution of defective organelles and may require studies of isolated tissues to fully define the presence of disease.

Slide 11: Clinical Utility of Blood Lactate-2

The circulating concentration of lactate and its clearance may also serve as a prognostic indicator and may eventually be useful as guides to treatment success. The examples above are two among many studies that support a role for lactate determination in the treatment of sepsis. The first study indicates that higher lactate concentrations are correlated with higher mortality rates. Concentrations above 4 mM exhibited the highest mortality. In addition, the utility of lactate concentrations was apparent in patients that presented with or without shock. The second study documented lactate concentrations in septic patients at presentation and 6 hours following. When lactate concentrations decreased greater than 10%, mortality was lower at both 30 and 60 days after presentation. While studies like these indicate the potential utility of lactate concentrations in guiding treatment, it remains unclear if lactate values are a better marker of the progression of sepsis than other clinical, physical, and laboratory indicators.

Slide 12: Laboratory Aspects of Lactic Acidosis-Preanalytic

As mentioned in Slide 10, lactate measurements are subject to a number of confounders. Many pre- analytic issues will increase apparent circulating lactate concentration. Muscle exertion such as fist clenching will result in increased local production of lactate and prolonged tourniquet application will slow diffusion and clearance of lactate. Glycolysis continues in vitro after blood sampling so high white blood cell counts, high temperatures, and long transport times will overestimate patient blood lactate. Collection tubes with glycolytic inhibitors such as fluoride may be employed if long transport times are unavoidable. Lactate concentrations are typically 10 fold higher than pyruvate in venous blood samples. Upon sample collection, pyruvate is rapidly converted to lactate as dictated by the equilibrium of the LDH reaction. Routine laboratory analysis of lactate, therefore, includes virtually all of the circulating pyruvate. Pyruvate may be preserved and analyzed only if the sample is treated immediately with a protein denaturing agent. Perchloric acid (5% final concentration) is commonly employed for this purpose.

Slide 13: Laboratory Aspects of Lactic Acidosis-Analytic

Circulating lactate is most commonly assessed in plasma using an enzymatic assay with LDH. Both endpoint and kinetic measurements have been employed that monitor the increase in NADH absorbance at 340 nm. Lactate determination in whole blood is performed on many blood gas systems using bacterial enzymes that oxidize lactate and produce hydrogen peroxide. Current resulting from oxidation of the peroxide is proportional to lactate concentration. Determination of lactate in whole blood must be performed rapidly to prevent in vitro production of lactate. The enzymes utilized in these techniques are generally very specific for the L-isomer of lactate. Interference from other short chain 2- hydroxyacids is not common. The amino acid alanine may also be used as a surrogate marker for lactate. Increased concentrations of lactate cause elevated concentrations of pyruvate by mass action. As the equilibrium constants of transaminases like ALT are near 1, elevated concentrations of pyruvate are reflected in elevated alanine concentrations.

Slide 14: Laboratory Aspects of Lactic Acidosis-Postanalytic

With proper sample procurement and transport, normal venous lactate concentrations range from 0.5-2.0 mM. Concentrations are transiently higher in neonates as lung function fully matures. Venous drainage collects the end products of metabolism and so venous lactate concentrations are higher than arterial concentrations. Due to differences in water content, plasma (water content » 0.93 kg/L) has a lactate concentration that is 5-10% higher than that of whole blood (water content » 0.84 kg/L). Due to imprecision and individual variability in measurements of Na, K, Cl, and CO2, mild lactate elevations (2-5 mM) may not cause an increased anion gap nor a significant decrease in pH.

Slide 15: Points to Remember

Lactate is an optically active 3-carbon hydroxycarboyxlic acid. Glycolysis produces two molecules of pyruvate. Pyruvate may be oxidized, carboxylated, or reduced to lactate. The latter process enables regeneration of glucose via the Cori cycle. Pyruvate oxidation and carboxylation is impeded when the capacity for cellular respiration is reduced, resulting in increased production of lactate. Cellular respiration may be impeded by a number of different mechanisms including hypoperfusion, toxins, or metabolic disorders. Determination of blood lactate concentration may be confounded by a number of pre-analytic factors including lactic acid generation at the site of phlebotomy and in vitro lactate production following sample acquisition. Increased blood lactate signals a broad array of diagnostic possibilities. In patients with sepsis/septic shock, blood lactate concentrations correlate with disease severity and survival.

Slide 16: References