Pilot study of mitochondrial bioenergetics in subjects with acute porphyrias

Abstract

Background and aims

The acute porphyrias are characterized by defects in heme synthesis, particularly in the liver. In some affected patients, there occurs a critical deficiency in a regulatory heme pool within hepatocytes that leads to up-regulation of 5-aminolevulinic acid [ALA] synthase-1, which is the first and normally rate-controlling enzyme in the pathway. In earlier work, we described defects in mitochondrial functions in cultured skin fibroblasts from patients with acute intermittent porphyria [AIP]. Others described defects in livers of murine models of AIP. Here, we explored mitochondrial energetics in peripheral blood mononuclear cells [PBMCs] and platelets in persons with AIP and hereditary coproporphyria [HCP]. Our hypotheses were that there are deficits in bioenergetic capacity in acute porphyrias and that subjects with more severe acute porphyria have more pronounced reductions in mitochondrial oxygen consumption rates [OCR].

Methods

We studied 17 subjects with acute hepatic porphyrias, 14 with classical AIP, one with severe AIP due to homozygous deficiency of hydroxymethylbilane synthase [HMBS], 2 with HCP, and 5 non-porphyric controls. We collected peripheral blood, isolated PBMCs, which we assayed either immediately or after frozen storage [80C] for up to 14 days. Using Seahorse XF-24-3, we measured OCR in the presence of glucose + pyruvate under basal condition, and after additions of oligomycin, carbonylcyanide p-trifluoromethoxyphenylhydrazone [FCCP], and antimycin+rotenone.

Results

Most subjects [13/17, 76%] were female. Subjects with moderate/severe symptoms associated with acute porphyria had significantly lower basal and maximal-OCR than those with no/mild symptoms who were the same as controls. We observed significant inverse correlation between urinary porphobilinogen [PBG] excretion and OCR. The subject with homozygous AIP had a much lower-OCR than his asymptomatic parents.

Summary/conclusions

Results support the hypothesis that active acute hepatic porphyria is characterized by a deficiency in mitochondrial function that is detectable in PBMCs, suggesting that limitations in electron transport and ATP production exist in such individuals.

Introduction

The porphyrias are a group of eight disorders, mainly due to inborn errors of metabolism, in which the primary defects are in normal heme synthesis. The principal sites of heme synthesis in vertebrates are the bone marrow (developing erythrocytes) and the liver (hepatocytes, which have high levels of cytochromes P-450), the principal users of hepatic heme. The human porphyrias generally are classified according to the principal site of overproduction of porphyrins or porphyrin precursors as being either hepatic or erythropoietic. The hepatic porphyrias are further classified as being “acute” or “inducible” or as being “chronic”. The acute designation is used for four disorders, all due to inherited defects in normal heme synthesis [acute intermittent porphyria, AIP; porphyria due to severe deficiency of ALA dehydratase; hereditary coproporphyria, HCP; and variegate porphyria]. In the USA, the most common and severe form is AIP, which is due to deficiency in activity of hydroxymethyl bilane synthase [HMBS, aka PBG deaminase], the third enzyme in the heme synthetic pathway. When AIP or other forms of acute porphyria are biochemically active, there is induction of 5-aminolevulinate [ALA] synthase-1, the first and normally rate-controlling enzyme of the heme biosynthetic pathway in the liver, leading to marked overproduction of ALA, and usually also of porphobilinogen [PBG]. [for reviews, see [[1], [2], [3]]].

The cardinal clinical features of symptomatic porphyrias are two-fold: [1] acute attacks of abdominal pain or pain in other parts of the body (chest, back, extremities), sometimes with the development of more diffuse neurological abnormalities, with weakness, loss of reflexes, cranial nerve involvement, delirium, and seizures; [2] cutaneous, with development of skin lesions on areas of skin exposed to sunlight.

Clinical manifestations of a porphyric attack are similar for each of the acute porphyrias [4], although usually more severe in AIP. Effects on the nervous system lead to most of the clinical features, including abdominal pain and neuro-visceral and circulatory disturbances. The extent of neurologic damage prior to correct diagnosis and therapy often determines the severity of the episode. Symptoms rarely occur prior to puberty and are more common and more severe in women, with peak incidence in the third and fourth decades of life. Certain drugs, excess alcohol ingestion, periods of fasting, emotional or physical stress or exhaustion, and the luteal phase of the menstrual cycle, when levels of progesterone peak, may trigger acute episodes.

The primary metabolic defect in AIP is reduction in activity of hepatic HMBS of about 50% due to various gene mutations that have been identified. Whether residual enzyme activity is sufficient for normal heme synthesis and functions of hemoproteins, such as mitochondrial and microsomal cytochromes, remains unclear. The defect in HMBS predisposes affected subjects to the deleterious influences of factors that may trigger acute attacks, which may occur during and likely due to up-regulation of the hepatic ALA synthase-1 gene. Such up-regulation may occur due to a primary effect on gene transcription (e.g., by action of nuclear factors) and/or due to increased demand for hepatic heme or decreased regulatory heme and thus to derivative up-regulation of ALA synthase-1 levels.

A defect in mitochondrial function as a factor in pathogenesis of the acute porphyrias was first proposed by Bonkovsky et al. Studies in cultured skin fibroblasts from AIP subjects showed ~ 50% decrease in activity of HMBS and a defect in mitochondrial NADH oxidation [5]. Recent studies performed in a murine model of AIP have shown that the mitochondrial respiratory chain [RC] and activity of the tricarboxylic acid [TCA] cycle were deficient in Hmbs −/− mice also treated with phenobarbital [6,7]. The authors proposed that marked induction of hepatic ALA synthase-1 led to shunting away of succinyl-CoA from the mitochondrial tricarboxylic acid [TCA] cycle into synthesis of ALA [cataplerosis]. There were major decreases in all respiratory complexes and enzymes of the mitochondria in livers, nerves, and muscles of the knockout mice. However, it is unclear to what extent studies in Hmbs −/− mice are a model relevant to human AIP: the mice require homozygous, rather than heterozygous deficiency of HMBS and they require further induction of ALA synthase-1 and cytochromes P-450 [Cyps] with phenobarbital.

Blood-based bioenergetic profiling has been successfully adopted as a reporter of systemic bioenergetic capacity in human studies [[1], [2], [3], [4], [5]]. For example, Dr. Molina's team has reported that the respiratory capacity of peripheral blood mononuclear cells (PBMCs) is related to key measures of age-related physical function decline [4,6]. More recently, studies of non-human primates conducted in Molina lab provide striking evidence that blood cells are able to recapitulate the bioenergetic capacities of highly metabolically active tissues such as skeletal muscle, cardiac muscle, and brain [7,8,9].

Because of our involvement in the US Porphyrias Consortium, we have been evaluating and following a sizable number of subjects of diverse demographic and clinical features with well-characterized acute hepatic porphyrias. We have had the opportunity to perform studies of oxygen consumption rates [using the Seahorse Instrument] in some of these subjects and to relate results to biochemical and genetic information that we collected and analyzed on the subjects. The aim of this study was to assess mitochondrial respiration in PBMCs of patients with acute hepatic porphyrias, compared to non-disease controls, and to assess whether defects in oxygen consumption rates correlate with clinical or biochemical features of disease. The main hypothesis that we tested is that subjects with biochemically active and clinically manifest AHP have significant defects in mitochondrial respiration that is detectable in PBMCs.