Peripheral lipoatrophy, central fat accumulation, hyperlipidaemia and insulin resistance, collectively referred to as lipodystrophy (LD) syndrome, commonly complicate therapy of human immunodeficiency virus (HIV) infection that includes a protease inhibitor [1–4]
Some physical features occur uncommonly in the absence of protease inhibitor therapy [1,3–7] Possible causes include other antiretroviral therapy and HIV infection. The major toxicities of nucleoside analogue reverse transcriptase inhibitor (NRTI) therapy are thought to be secondary to mitochondrial DNA polymerase gamma inhibition resulting in impaired synthesis of mitochondrial enzymes that generate ATP  These include anaemia and myopathy (zidovudine), neuropathy (stavudine, didanosine, zalcitabine), pancreatitis (didanosine) and hepatic steatosis and lactic acidaemia (fialuridine, didanosine, stavudine, zidovudine). Hepatic failure and death have occurred rarely but few reports mention associated wasting.
Fourteen patients receiving combination NRTI therapy but having never been treated with a protease inhibitor presented with clinical features consistent with LD syndrome and most were found to have lactic acidaemia and liver dysfunction. This study was undertaken to determine the relationship between lipoatrophy and lactic acidaemia in NRTI recipients, to describe clinical and metabolic features and possible risk factors, to identify distinguishing features from protease inhibitor-related LD syndrome and if NRTI therapy contributes to any LD syndrome component in patients receiving combination NRTI-protease inhibitor therapy.
Cases comprised all patients with lipoatrophy with or without abdominal distension or buffalo hump identified between May 1998 and January 1999, who were receiving NRTI therapy but had never received a protease inhibitor (n = 14). The controls comprised antiretroviral-naive patients without LD syndrome (n = 32), NRTI recipients without LD syndrome (n = 28), NRTI-protease inhibitor recipients without LD syndrome (n = 44), and lipodystrophic NRTI-protease inhibitor recipients (n = 102). The controls were all healthy outpatients who were seen for routine care between October 1998 and February 1999, recruited to the Australian lipodystrophy prevalence survey, matched for sex (all male, a parameter that strongly affects body composition) and not receiving steroid or lipid-lowering therapy. No patient had an AIDS-defining condition in the preceding 3 months. Cases were studied to February 1999. The study was approved by the hospital's Research Ethics Committee and all subjects provided written, informed consent.
LD syndrome was defined by patient report (standardized questionnaire) of peripheral lipoatrophy (fat loss from face, arms, buttocks or legs) and/or central fat accumulation (abdomen, dorso-cervical fat pad) confirmed by physical examination  Liver involvement in cases was defined by the presence of one or more of hepatomegaly, ascites, peripheral oedema or encephalopathy.
Age, HIV duration, presence of AIDS, types and durations of all antiretroviral therapies, and weight were recorded. Symptoms and signs were recorded for cases and those that were common during the preceding 3 months (fatigue, nausea, weight loss of at least 3 kg) were evaluated in controls. CD4 count, HIV RNA load, electrolytes, liver enzymes, and lipid and glycaemic parameters were determined as previously described [1,4] Prior weights, anion gaps and liver enzymes were recorded where available. Lactate was measured by the Vitros enzymatic method (Johnson and Johnson Clinical Diagnostics, Rochester, New York, USA). Lactic acidaemia was defined as > 2.0 mmol/l and anion gap as sodium plus potassium minus (chloride plus bicarbonate)  Beta-hydroxybutyrate was measured in six cases in order to exclude ketoacidosis. Body composition was measured by dual-energy X-ray absorptiometry (DEXA; Lunar DPXL, Madison, Wisconsin, USA) [1,4] Intra-abdominal and extra-abdominal fat at the L4 vertebral level was estimated by single cut computerized tomography  Six cases had whole abdominal computerized tomography performed because of abdominal distension or ascites.
Comparisons between groups used the Mann–Whitney test for continuous variables and Fisher's exact test for categorical variables. Risk factors for NRTI lipoatrophy were assessed using a logistic regression based on cases and NRTI controls. Factors assessed were age, AIDS status, pre-therapy weight, current weight, CD4 count and viral load and type and duration of therapy. Logistic regression analyses of treated controls evaluated the impact of clinical parameters and lactic acidaemia on the presence of peripheral lipoatrophy, abdominal obesity and buffalo hump. Similar analyses based on protease inhibitor controls evaluated the impact of each protease inhibitor regimen.
Data for cases and controls are shown in Table 1. Two cases and 11 controls were also receiving abacavir. Protease inhibitor therapy comprised indinavir (n = 79), ritonavir-saquinavir (n = 28), nelfinavir (n = 19), nelfinavir-saquinavir (n = 10) and saquinavir (n = 10). There was no significant difference in symptom prevalence, body composition or metabolic parameters between untreated and NRTI controls. All groups had similar weight pre-therapy, a parameter associated with subsequent severity of LD syndrome 
The cases were more likely to be receiving stavudine with or without hydroxyurea than NRTI controls. Ten (71%) cases were receiving stavudine-didanosine versus seven (25%) NRTI controls (P = 0.003); all but two cases had received zidovudine. The mean current NRTI therapy duration was 14 months. Factors independently associated with lipoatrophy in NRTI-only recipients were current stavudine use, greater NRTI-therapy duration and increasing age (Table 2).
The main clinical features in cases were recent onset fatigue, nausea and weight loss (mean, 6 kg; range, 2 to 14 kg), lipoatrophy of the face, legs (both n = 14), buttocks (n = 11) and arms (n = 10), abdominal distension (n = 7) and non-tender, smooth hepatomegaly (n = 6). No patient had other organomegaly or intestinal obstruction, although three had ascites. Other features were ankle oedema, dyspnoea (both n = 3), buffalo hump, gynaecomastia (both n = 2) and hepatic encephalopathy (n = 1). Three cases had pre-existing, mild peripheral neuropathy which was stable at diagnosis; none had jaundice, muscle wasting or anaemia. The mean duration of constitutional symptoms was 4 months (range, 0 to 9 months). The clinical intensity varied: three cases were hospitalized (for 3, 4 and 13 weeks), and four patients continue therapy because of milder, stable clinical features.
Cases had significantly less limb and subcutaneous abdominal fat than untreated and NRTI controls without lipoatrophy and greater intra-abdominal fat than untreated controls (Table 1), but similar total and regional muscle (data not shown). Cases with liver involvement had similar body composition to those without (data not shown). Computerized tomography in six cases with abdominal distension demonstrated probable hepatic steatosis in five cases as well as the ascites in three cases but no intestinal obstruction or other organomegaly. No factor such as alcohol abuse, active viral hepatitis, auto-immune hepatitis, hepatic mass or biliary obstruction was identified as a cause of liver dysfunction. Liver biopsy was not performed in the most affected cases because of coagulopathy. Ascitic fluid in one case was acellular.
Cases had higher mean lactate than controls. Causes of lactic acidaemia such as hypoperfusion, hypoxia, renal failure or relevant drugs, were not evident  Lactate correlated with change in body weight on therapy in cases (r2 = 0.64; P = 0.001) but not in control groups (r2 < 0.0001 to 0.16). Lactate was higher in the seven cases with liver involvement than in cases without liver involvement (6.8 and 2.7 mmol/l, respectively; P = 0.02) who in turn had higher lactate than NRTI controls (P = 0.03). Cases had greater anion gap and hepatic enzymes and lower bicarbonate, chloride and albumin than controls; there was no significant difference for sodium or potassium, and beta-hydroxybutyrate was not elevated (data not shown).
Liver enzymes and anion gap increased in most cases over the 3 to 6 months prior to diagnosis (Fig. 1) but did not appear to precede onset of symptoms or weight loss. No liver enzyme value was of grade 4 severity. Cases with ascites had mildly elevated prothrombin time but normal arterial pH (data not shown). C-peptide was the only lipid or glycaemic parameter that differed significantly between cases and both untreated and NRTI controls.
Regardless of group, patients with lactic acidaemia were older than patients with normal lactate (44.9 and 42.2 years, respectively; P = 0.04), had received more stavudine (17 and 12 months; P = 0.02), more didanosine (9 and 7 months; P = 0.01), and were more likely to have recent onset weight loss (40 and 9%; P < 0.0001), fatigue (58 and 15%; P < 0.0001) and nausea (40 and 10%; P < 0.0001). Protease inhibitor controls with lipoatrophy were more likely to have lactic acidaemia than those without lipoatrophy, although this difference was not significant (P = 0.10).
Withdrawal of therapy
Ten cases ceased therapy. One case with lactate of 13.2 mmol/l and prior AIDS dementia died after 3 months of progressive encephalopathy and wasting. The other nine cases (mean lactate, 4.5 mmol/l; range, 2.8 to 9.0 mmol/l) were followed for an average of 4 months (range, 1 to 9 months). Fatigue and nausea generally improved but four cases remained symptomatic. Weight gain was limited (mean, 2.5 kg; Fig. 1) and only two cases reached their pre-therapy weight. Ascites required frusemide and spironolactone for 9 months in one case. Lactic acidaemia resolved in seven cases in a mean time period of 3 months after ceasing therapy; anion gap and liver enzymes also fell. HIV RNA load rose and CD4 counts decreased (data not shown) but no patient developed an HIV-related opportunistic condition. The four cases (mean lactate, 2.3 mmol/l; range, 0.9 to 3.2 mmol/l) who remained on NRTI therapy had stable hepatic and metabolic parameters over an average of 2 months (range, 1 to 3 months) of follow-up.
Six NRTI-protease inhibitor lipodystrophic controls, but no other control, also ceased NRTI therapy because of lactic acidaemia (mean, 3.3 mmol/l) and weight loss, nausea or fatigue.
Relationship to protease inhibitor-related LD
No physical or body composition parameter distinguished patients with NRTI-LD syndrome from those with protease inhibitor-related LD syndrome, including the regional prevalences of lipoatrophy (data not shown). Presence of recent onset symptoms and weight loss, and numerous metabolic parameters distinguished cases from protease inhibitor-related LD syndrome (Table 1).
Factors associated with peripheral lipoatrophy, abdominal obesity and buffalo hump in treated controls are shown in Table 3. All protease inhibitor regimens were associated with similar risks of lipoatrophy, abdominal obesity and buffalo hump except hard-gel saquinavir alone (odds ratio for peripheral lipoatrophy as compared with patients receiving indinavir, 0.02; 95% confidence interval, 0.001 to 0.21; P = 0.002 after adjustment for current stavudine use, total NRTI duration and presence of lactic acidaemia).
NRTI therapy can cause a partially reversible syndrome of subacute onset fatigue, nausea, peripheral lipoatrophy, abdominal distension, lactic acidaemia and hepatic dysfunction. Symptoms and liver dysfunction are more likely with higher lactate but lipoatrophy can occur with lower and even normal lactate. Both NRTI therapy and protease inhibitor therapy appear to be associated with the physical features of LD syndrome, but NRTI and protease inhibitor therapy induce distinguishing symptoms and metabolic features that might assist in attributing aetiology in a given patient.
Metabolic disturbances, nausea and fatigue mostly resolved after NRTI cessation but weight recovery was limited, an outcome similar to that seen with known NRTI-induced mitochondrial toxicities  Longer follow-up is clearly required. Riboflavin, thiamine, co-enzyme Q, dichloroacetate and carnitine are rarely beneficial in congenital lactic acidaemia as they usually affect oxidation upstream of the defect  Nevertheless, they merit evaluation as an alternative to NRTI cessation and for primary prevention.
All physical features of LD syndrome were associated with use and duration of both protease inhibitor therapy and at least one NRTI, particularly in the presence of lactic acidaemia. Imaging did not reveal a definite increase in intra-abdominal fat in cases, and it may be that some abdominal distension observed was due to clinically undiagnosed ascites rather than fat accumulation. The association of lipoatrophy with current stavudine therapy and the apparent weak protective effect of zidovudine therapy should be interpreted with caution; zidovudine and stavudine are never prescribed together because of intracellular antagonism, zidovudine was licensed years before stavudine and so most cases receiving stavudine had previously received zidovudine. Why lamivudine was associated with abdominal distension is unclear; lamivudine has not been shown to affect hepatic mitochondria  Any role of hydroxyurea needs investigation; although hydroxyurea effectively increases intra-cellular NRTI-triphosphate concentrations, chronic hydroxyurea therapy does not induce lipoatrophy in patients with sickle cell anemia (who of course do not receive NRTI therapy) [11,12] Randomized studies will be required to confirm these preliminary data and to further define the relative contributions of NRTI and protease inhibitor therapy to each component of the syndrome. The association of NRTI-related lipoatrophy with greater age concurs with data demonstrating accumulation of mitochondrial genomic defects with age 
The strongest metabolic predictor of future LD severity in NRTI-protease inhibitor recipients was elevated serum C-peptide  In the present study, C-peptide was the only metabolic parameter elevated with both NRTI and protease inhibitor therapies. These data support our interpretation of the present findings that both NRTI and protease inhibitor therapy can cause LD syndrome.
The pathogenesis of NRTI-related LD syndrome is unknown. The association of lipoatrophy and abdominal distension with lactic acidaemia suggests these features represent NRTI mitochondrial toxicity. Indeed, mutations in mitochondrial DNA polymerase gamma can be associated with a similar syndrome, multiple symmetric lipomatosis  Alternatively, primary selective impairment of hepatic glycogen and fat oxidation might result in increased oxidation of peripheral energy stores and so to lipoatrophy. Weight loss could result from hepatitis-induced nausea and anorexia but cases without liver impairment still had lipoatrophy, and preferential peripheral fat loss with reduced caloric intake is unusual 
Our study could not determine the prevalence of NRTI LD syndrome, whether lactic acidaemia precedes (ie predicts) this syndrome, whether increases in lactate within the reference range can be associated with LD syndrome nor if factors such as pre-existing liver disease predispose to lipoatrophy. All patients were male and only 13 patients were receiving the newer NRTI, abacavir. It remains unknown if alternative NRTIs can be safely administered to affected patients. Lastly, triple NRTI therapy is one recommended option for HIV therapy  its potential for inducing lipoatrophy and lactic acidaemia needs urgent assessment.
It may be useful to monitor lactate in NRTI recipients, particularly those who develop lipoatrophy, constitutional illness or liver failure. These features in the presence of lactic acidaemia may necessitate NRTI cessation if other causes are excluded, but preferably only if safe and effective alternative antiretroviral therapy is available or the risk of HIV disease progression with no therapy is small. Lipoatrophy in NRTI-protease inhibitor recipients should not lead to change of protease inhibitor therapy without lactate, lipids and glycaemic parameters being measured. Therapies that will allow for NRTI therapy to be continued in the presence of lactic acidaemia are required, as are newer less toxic NRTI agents.
To the patients who participated in the study; to Drs Timothy Barnes, Deborah Marriott, Ross Price and Dick Quan for patient referral; to Donald Chisholm, Graham Jones and J. Dennis McGarry for helpful comments.
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